CA2516652A1 - System for external control of oncolytic virus replication - Google Patents

Abstract

Compositions, methods and a system for external regulation of oncolytic virus replication are provided. The oncolytic virus contains a cell type-specific transcriptional regulatory element (CT-TRE) and an inducible transactivator regulated transcriptional regulatory element. The virus preferentially replicates in cells that allow a CT-TRE to function. In addition, the concentration of an inducing agent or condition is used to regulate a transactivator regulated transcriptional regulatory element, thereby providing at least two levels of control of oncolytic viral replication.

Description

SYSTEM FOR EXTERNAL CONTROL OF ONCOLYTIC VIRUS REPLICATION
Background Gene therapy forms the basis of innovative and potentially powerFul disease-fighting tools, in which an exogenous nucleotide is provided to a cell. This approach holds great potential in treating not only cancer, but many other diseases as well, including cystic fibrosis, anemia, hemophilia, diabetes, Hungtington's disease, AIDS, abnormally high serum cholesterol levels, certain immune deficiencies, and many forms of cancer.
Gene therapy generally requires a delivery vehicle for the exogenous sequence, such as a viral or non viral vector. A variety of viral vectors have shown therapeutic efficacy against these diseases. For reviews, see e.g., Sandrin V et al., Curr Top Microbiol Immunol.
2003;281:137-78; Buning H., Gene Ther. 2003 Ju1;10(14):1142-51 and St George JA, Gene Ther. 2003 Ju1;10(14):1135-41.
In an alternate approach applicable to cancer treatment, specific attenuated replication-competent viral vectors have been developed in which selective replication in cancer cells preferentially destroys those cells. For example, various cell-specific replication-competent adenovirus constructs, which preferentially replicate (and thus destroy) certain cell types, are described in, for example, WO 95/19434, WO
98/39465, WO
98/39457, WO 98/394.66, WO 99/06576, WO 98/39464, WO 00/15820.
Factors that make adenovirus a safe therapeutic agent include the facts that:
(a) infection with adenovirus has minor clinical disease manifestations; (b) adenovirus has a stable well-described and characterized genome; (c) adenovirus typically does not integrate its viral DNA into host DNA; (d) adenovirus infection results in transient gene expression; (e) adenovirus is able to infect both dividing and non-dividing cells; (~
adenovirus can infect a variety of human cell types; (g) adenovirus is physically stable; and (h) adenovirus is amenable to high titer production.
A continuing concern regarding gene therapy in patients is potential toxicity.
It follows that the ability to regulate virus infection and/or gene expression in vivo wquld be advantageous. Regulated gene expression for purposes of gene therapy is reviewed, for example, by ~oltick and Wilson (2001 ) Ann NY Acad Sci. 953:53-63; Harrington et al.
$,(2000) Adv Drug Deliv Rev. 44(2-3):167-84; and Clackson (2000) Gene Ther.
7(2):120-5.
Unlike replication defective vectors which are typically used for gene therapy, oncolytic vectors such as those exemplified herein, replicate in vivo producing progeny which infect additional target cells. It follows that there is substantial interest in being able to regulate the replication of oncolytic vectors in vivo.

Summary Of The Invention Replication-competent viral vectors, and methods for their use are provided.
The invention provides compositions comprising replication pompetent viruses having at least one viral gene under transcriptional control of a regulated gene expression sysfiem, as exemplified herein by replication competent adenovirus. The regulated gene expression system comprises at least two levels of transcriptional regulation. At a first level of regulation, a transcriptional transactivator coding sequence is under the transcriptional control of a cell type-specific transcriptional response element (CT-TRE). At a second level of regulation, a viral gene is under the transcriptional control regulated by interaction of the transeriptional transactivator with a transcriptional response element (referred to herein as a transcriptional transactivator regulated response element. Transactivators of interest in practicing the invention functionally interact with an inducing agent, usually a non-native inducing agent which is exogenously provided to the host cells of interest, e.g. a chemical entity such as tetracycline, ecdysone, rapamycin, synthetic progesterones, glucocorticoids, or an analog thereof, etc. The inducing agent may also be a non-chemical inducer, i.e.
ultra-sound, heat, external beam radiation; hypoxia, etc.
The transcriptional transactivator is only produced in cells where the cell type-specific THE is active. The activity of the transcriptional transactivator is dependent upon the presence/concentration of a particular inducing agent or condition. The inducing agent or condition therefore provides a means to regulate the function of the transcriptional transactivator. In the presence of the appropriate concentration of inducing agent an/or condition, an activating transcriptional transactivator will activate transcription and an inhibitory transcriptional transactivator will inhibit transcription.
Expression of a gene essential for viral replication is thus regulated, thereby regulating virus replication and spread. Hence, the replication and spread of an oncolytic virus may be regulated in vitro or in vivo by providing the inducing agent and/or adjusting the inducing agent concentration using the compositions and methods described herein.
Brief Description Of The Drawings Figure ~ is a schematic illustrating the genome of a prostate specific adenovirus vector with tetracycline regulated replication control.
Figure 2 is a schematic illustrating the genome of a prostate specific adenovirus vector armed with GM-CSF, with tetracycline regulated replication and transgene control.
Figure 3 is a schematic illustrating the genome of an adenovirus vector with dual tetracycline regulated replication control.
Figure 4 is a schematic illustrating the genome of an adenovirus vector with rapamycin (ARIAD system) regulated control for use in pan-cancer applications.

Detailed Description of the Embodiments Regulated replication-competent viral vectors are provided, which comprise a cell type-specific transcriptional regulatory element (CT-TRE) and a transactivator regulated transcriptional regulatory element, which is inducible. The virus preferentially replicates in cells that allow function of the CT-TRE, and based on the presence or absence of an inducer that regulates the transactivator regulated transcriptional regulatory element.
The CT-THE controls transcription of an inducible transcriptional transactivator (TA) coding sequence. The transcriptional transactivator (a) requires an inducing agent to be functional and (b) controls transcription of a viral gene. The inducer is preferably an exogenous compound, not normally present in the host cells of interest, e.g.
tetracycline, ecdysone, rapamycin, a synthetic progesterone, a glucocorticoid, and the lilts, including analogs thereof, or the inducer may be ultra-sound, heat, ultra-sound, heat, external beam radiation, hypoxia or some other treatment.
An inhibitory transcriptional transactivator will inhibit transcription in the presence of the inducing agent, and an activating transcriptional transactivator will activate transcription in the presence of the inducing agent. In this way, expression of a viral gene essential for replication is regulated both by the CT-THE and the transactivator regulated transcriptional regulatory element, and indirectly by the concentration of the inducing agent or condition. It follows that during treatment of a patient with oncolytic virus therapy, replication and spread of the virus may be regulated by adjusting the inducing agent concentration.
Depending on the specific uses of the viral vector, the inducing agent may act to inhibit transcription, or to enhance transcription. Transcription inhibitors provide a way to "shut down" viral replication by delivering the inducing agent to the host cells. Transcription activators provide a way to induce viral replicati~n by addition of the inducing agent.
Preferably the viral gene essential for replication is an early gene. In some embodiments, the viral vectors of this invention comprise a viral gene under the transcriptional control of a transactivator regulated transcriptional regulatory element, and at least one other gene, such as an additional viral gene or a transgene, under control of either the same transactivator regulated transcriptional regulatory element or a second transactivator regulated transcriptional regulatory element that is substantially identical to the first transactivator regulated transcriptional regulatory element.
Preferably, the first and second genes under transcriptional control of the transactivator regulated transcriptional regulatory elements) are both viral genes necessary for replication. By providing for cell-specific transcription through the use of one or more transactivator regulated transcriptional regulatory elements, the invention provides viral vectors that can be used for cell-specific replication resulting in selective cytolysis of target cells, where viral replication is regulated through exposure of the target cells to an exogenous inducing agent or condition.

The viral vectors of the invention replicate preferentially in CT-THE
functional cells, referred to herein as target cells. This replication preference is indicated by comparing the level of replication (i.e., titer) in cells in which the CT-THE is active to the level of replication in cells in which the CT-THE is not active (i.e., a non-target cell).
Comparison of the viral titer following infection of a target cell to the titer following infection of a non-target cell (CT-TRE inactive) provides a key indication that the overall replication preference is enhanced due to the replication in target cells and depressed replication in non-target cells. This provides a means for targeted cell killing, such that runaway infection is prevented. A further level of replication control is provided by the presence or absence of an inducing agent or condition or changes in the concentration thereof. The viral vectors of the invention are exemplified herein by replication competent adenovirus which exhibit target cell specific replication, the control of which is further regulated by an inducing agent.
General Technidues The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biol~gy (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are within the skill of the art.
Such techniques are explained fully in the literature, such as, "Molecular Cloning: A
Laboratory Manual", second edition (Sambrook et al., 199); "~ligonucleotide Synthesis"
(M.J. Gait, ed., 194.); "Animal Cell Culture" (R.I. Freshney, ed., 197);
"Methods in Enzymology" (Academic Press, Inc.); "Handbook of Experimental Immunology"
(D.M. Weir &~ C.C. Blackwell, eds.); "Gene Transfer ~/ectors for Mammalian Cells" (J.M.
Miller ~ M.P.
Calos, eds., 197); "Current Protocols in Molecular Biology" (F.M. Ausubel et al., eds., 19137); "PCR: The Polymerase Chain Reaction", (Mullis et al., eds., 1994); and "Current Protocols in Immunology" (J.E. Coligan et al., eds., 1991).
Definitions A "replication competent" or "oncolytic" vector of the present invention (used interchangeably herein) can be in any of several forms, including, but not limited to, naked DNA; a viral vector encapsulated in a virus coat; a viral vector packaged in another viral or viral-like form (such as herpes simplex virus and AAV); a viral vector encapsulated in a liposome; a viral vector complexed with polylysine or other biocompatible polymer; a viral vector complexed with synthetic polycationic molecules; a viral vector conjugated with transferrin; a viral vector complexed with compounds such as PEG to immunologically "mask" the molecule and/or increase half-life; or a viral vector conjugated to a non-viral protein or any delivery vehicle known to those of skill in the art. Such vectors represent one v embodiment of the invention. Preferably, the polynucleotide is DNA. As used herein, "DNA" includes not only bases A, T, C, and G, but also includes any of their analogs or modified forms of these bases, such as methylated nucleotides, internucleotide modifications such as uncharged linkages and thioates, use of sugar analogs, and modified and/or alternative backbone structures, such as polyamides. For purposes of this invention, viral vectors are replication-competent in a target cell. Exemplary replication-competent viruses included within the scope of the invention include, but are not limited to, adenoviruses, vesicular stomatitis viruses (VSV), herpes viruses (e.g., herpes simplex virus;
HSV), reoviruses, paramyxoviruses, rhinoviruses, Newcastle disease viruses, polioviruses, West Nile virus, coxsackie virus, measles viruses and vaccinia viruses, etc.
Any and all serotypes of replication-competent viruses can be engineered for targeted expression and regulated expression using the techniques described herein.
An "adenovirus vector" or "adenoviral vector" (used interchangeably herein) is a term well understood in the art and generally comprises a polynucleotide (defined herein) including all or a portion of an adenovirus genome. As used herein, "adenovirus" refers to the virus itself or derivatives thereof. The term covers all serotypes and subtypes and both naturally occurring and recombinant forms, except where otherwise indicated.
For the purposes of the present invention, regulatable adenovirus vectors are provided which contain a cell type-specific transcriptional regulatory element (CT-TRE) operably linked to an inducible transactivator gene, and an adenovirus gene operably linked to a transcriptional transactivator regulated regulatory element regulated by the inducible transactivator. The adenovirus vector may optionally contain a second adenoviral gene or a transgene operably linked to a transactivator regulated regulatory element or another type of transcriptional regulatory element, which is not cell type-specific. For techniques related specifically to adenovirus, see, inter alia, Felgner and Ringold (1989) Nafure 337:387-388;
Berkner and Sharp (1983) Nucl. Acids Res. 11:6003-6020; Graham (1984) EMB~ J.
3:2917-2922; Bett et al. (1993) J. Virology 67:5911-5921; Bett et al. (1994) Proc. Natl.
Acad. Sei. USA 91:8802-8806. Publications describing various aspects of adenovirus biology and/or techniques relating to adenovirus include the following. Graham and Van de Eb (1973) Virology 52:456-467; Takiff et al. (1981) Lancet ii:832-834; Berkner and Sharp (1983) Nucleic Acid Research 6003-6020; Graham (1984) EMB~ J 3:2917-2922; Bett et al.
(1993) J. Virology67:5911-5921; and Bett et al. (1994) Proc. Natl. Acad. Sci.
USA 91:8802-8806 describe replication-defective adenoviruses that have been genetically modified to act as gene transfer vehicles (i.e., gene therapy). In such vectors, typically the early adenovirus gene products E1A and E1 B are deleted and provided in trans by the packaging cell line 293 developed by Frank Graham (Graham et al. (1987) J. Gen. Birol. 36:59-72 and Graham (1977) J. Genetic Virology 68:937-940). The gene to be transduced is commonly inserted into adenovirus in the deleted E1A and E1 B region of the virus genome Bett et al. (1994), supra. Adenovirus vectors for gene therapy have been described by Stratford-Perricaudet (1990) Human Gene Therapy 1:2-256; Rosenfeld (1991) Science 252:431-434; Wang et al.
(1991) Adv. Exp. Med. Biol. 309:61-66; Jaffe et al. (1992) Nat Gent. 1:372-378; Quantin et al. (1992) Proc Natl. Acad. Sci. USA 89:2581-2584; Rosenfeld et al. (1992) Cell 68:143-155; Stratford-Perricaudet et al. (1992) J. Clin. Invest. 90:626-630; Le Gal La Salle et al.
(1993) Science 259:988-990; Mastrangeli et al. (1993) J. Clin. Invest. 91:225-234; Ragot et al. (1993) Nature 361:647-650; Hayaski et al. (1994) J. BioL Chem. 269:23872-23875.
By "transactivator," "transactivating factor," or "transcriptional activator"
is meant a polypeptide that facilitates transcription from a promoter. Inducible transactivators facilitate transcription in the presence (or absence) or due to a change in concentration of a specific inducing agent or condition. For example, a tet regulated inducible transactivator may facilitate transcription from the inducible tet~ promoter when the transactivator is not bound to the appropriate inducer, e.g., tetracycline or an analog thereof. In presence of tetracycline, such a transactivator is prevented from binding to its target and thus transcription is blocked. A reverse tet transactivator retains the DNA binding specificity of a wild-type tet repressor, but is regulated in a reverse manner, i.e. it binds to a tet operator sequence only in the presence of the appropriate inducer, e.g., tetracycline or an analog thereof, rather than in the absence of the inducer. Transcriptional activators generally bind directly to a transcriptional response element, however in some cases they bind indirectly to another protein, which in turn binds to or is bound to the transcriptional response element.
As used herein, a "transcriptional regulatory element", also referred to as a "transcriptional response element" or "TRE" is a polynucleotide sequence, preferably a DNA
sequence, that regulates (i.e., controls) transcription of an operably-linked polynucleofiide sequence by an RNA polymerase to form RNA. As used herein, a THE increases transcription of an operably linked polynucleotide sequence in a host cell that allows the THE to function. The THE comprises an enhancer element and/or promoter element, which may or may not be derived from the same gene. The promoter and enhancer components of a THE may be in any orientation and/or distance from the coding sequence of interest, and may comprise multimers of the foregoing, as long as the desired transcriptional activity is obtained. As discussed herein, a THE may or may not lack a silencer element. A THE
may be cell type specific, e.g., specific to cancer cells derived from any of a variety of tissue types (cell status specific), prostate cancer cells, bladder cancer cells, colon cancer cells, liver cancer cells, kidney cancer cells, breast cancer cells, pancreatic cancer cells, etc.) or may be active in a large number of cell types. As used herein, the term cell type-specific transcriptional regulatory element (CT-TRE) refers to both cell type specific and cell status specific regulatory elements.

The transcriptional regulatory element upon which the transactivator acts is referred to as a "transcriptional transactivator regulated regulatory element". A
transcriptional transactivator regulated regulatory element regulates transcription of an operably-linked polynucleotide sequence, and is regulated by an inducible transactivator, for example by binding of an inducible transactivator protein to a specific DNA binding site within or near the promoter. As used herein, a transcriptional transactivator regulated regulatory element or TA-THE alters transcription levels in the presence of the inducing agent, either by downregulating or upregulating transcription.
An "enhancer" is a term well understood in the art and is a nucleotide sequence derived from a gene which increases transcription of a gene that is operably-linked to a promoter to an extent which is greater than the transcription activation effected by the promoter in the absence of the enhancer when operably-linked to the gene, i.e.
it increases transcription from the promoter. Having "enhancer activity" is a term well understood in the art and means that when present the nucleotide sequence which has "enhancer activity"
increases transcription of a gene which is operably linked to a promoter to a level which is greater than the level of transcription effected by the promoter itself when operably linked to the gene in the absence of the nucleotide sequence which has "enhancer activity", i.e., it increases transcription from the promoter.
"Under transcriptional control" is a term well-understood in the art and indicates that transcription of a polynucleotide sequence, usually a DNA sequence, depends on its being operably (operatively) linked to an element that contributes to the regulation of, either promotes or inhibits, transcription.
The term "operably linked" relates to the orientation of polynucleotide elements in a functional relationship. A THE is operably linked to a coding segment if the THE promotes transcription of the coding sequence. ~perably linked means that the DNA
sequences being linked are generally contiguous and, where necessary to join two protein coding regions, contiguous and in the same reading frame. However, since enhancers generally function when separated from the promoter by several kilobases and intronic sequences may be of variable length, some polynucleotide elements may be operably linked but not contiguous.
The terms "inducing agent" and "inducing condition" may be used interchangeably and refer to a chemical entity or condition which facilitates binding of an inducible transactivator transcriptional response element to a specific DNA binding site within or near a promoter. As used herein, when the inducing agent/condition acts on a transactivator transcriptional response element, transcription levels are altered or modulated, either by downregulating or upregulating transcription. Exemplary "inducing agents" and "inducing conditions" include, but are not limited to chemical or non-chemical entities (such as ultra-sound, heat, external beam radiation, hypoxia, etc.). It will be understood that exposure of an oncolytic vector of the invention to such an "inducing agent" or "inducing condition may result in either activation (induction) or suppression (repression) of transcription, resulting in an increase or decrease in viral replication, respectively.
A "host cell" includes an individual cell or cell culture which can be or has been a recipient of any virus and/or vector of the present invention. Host cells include progeny of a single host cell, and the progeny may not necessarily be completely identical (in morphology or in terms of total DNA complement) to the original parent cell due to natural, accidental, or deliberate mutation and/or change. A host cell includes cells transfected or infected in viv~ with a virus and/or vector of the invention. Host cells may be isolated from a tissue or animal host for in vitr~ culture.
As used herein, the terms "neoplastic cells", "neoplasia", "tumor", "tumor cells", "cancer" and "cancer cells", (used interchangeably herein) refer to cells which exhibit relatively autonomous growth, so that they exhibit an aberrant growth phenotype characterized by a significant loss of control of cell proliferation.
Neoplastic cells can be malignant or benign.
In the context of a viral vector, e.g., the viral vector(s), of the invention, a "heterologous" promoter or enhancer is one that is not present in wild-type virus. Examples of a heterologous promoter or enhancer are the albumin promoter or enhancer and other viral promoters and enhancers, such as SV40.
In the context of viruses (viral vectors), an "endogenous" promoter, enhancer, or THE is native to, or derived from, the virus.
The term "gene" is well understood in the art and is a polynucleotide encoding a polypeptide. In addition to the polypeptide coding regions, a gene includes non-coding regions including, but not limited to, introns, transcribed but untranslated segments, and regulatory elements upstream and downstream of the coding segments.
In the context of viruses (viral vectors), a "heterologous polynucleotide" or "transgene" is any gene that is not present in wild-type virus. Preferably, the transgene will also not be expressed or present in the target cell prior to introduction by the virus (viral vector). Examples of preferred transgenes are provided below.
A sequence, whether polynucleotide or polypeptide, "depicted in" a SEQ ID NO, means that the sequence is present as an identical contiguous sequence in the sequence of the SEQ ID NO.
The terms "polynucleotide" and "nucleic acid", used interchangeably herein, refer to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. These terms include a single-, double- or triple-stranded DNA, genomic DNA, cDNA, RNA, DNA-RNA hybrid, or a polymer comprising purine and pyrimidine bases, or other natural, chemically, biochemically modified, non-natural or derivatized nucleotide bases. The following are non-limiting examples of polynucleotides: a gene or gene fragment, exons, introns, mRNA, tRNA, rRNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. A
polynucleotide may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs, uracyl, other sugars and linking groups such as fluororibose and thioate, and nucleotide branches. The sequence of nucleotides may be interrupted by non-nucleotide components. A polynucleotide may be further modified after polymerization, such as by conjugation with a labeling component. ~ther types of modifications included in this definition are caps, substitution of one or more of the naturally occurring nucleotides with an analog, and introduction of means for attaching the polynucleotide to proteins, metal ions, labeling components, other polynucleotides, or a solid support.
Preferably, the polynucleotide is DNA. As used herein, "DNA" includes not only bases A, T, C, and G, but also includes any of their analogs or modified forms of these bases, such as methylated nucleotides, internucleotide modifications such as uncharged linkages and thioates, use of sugar analogs, and modified and/or alternative backbone structures, such as polyamides.
A polynucleotide or polynucleotide region which has a certain percentage (for example, 30%, 85%, 90%, or 95%) "sequence identity" to another sequence means that, when aligned, that percentage of bases are the same in comparing the two sequences.
This alignment and the percent homology or sequence identity can be determined using software programs known in the ark, for example those described in Current Proloc~Is in 11~~lecular biology (F.NI. Ausubel et al., eds. 1987) Supplement 30, section 7.7.13, Table 7.7.1. A preferred alignment program is ALIGN Plus (Scientific and Educational Software, Pennsylvania), preferably using default parameters.
The terms "polypeptide", "peptide" and "protein" are used interchangeably to refer to polymers of amino acids of any length. These terms also include proteins that are post-translationally modified through reactions that include glycosylation, acetylation and phosphorylation.
"Replication" and "propagation" are used interchangeably and refer to the ability of a viral vector of the invention to reproduce or proliferate. This term is well understood in the art. For purposes of this invention, replication involves production of viral proteins and is generally directed to reproduction of virus. Replication can be measured using assays standard in the art and described herein, such as a burst assay or plaque assay.
"Replication" and "propagation" include any activity directly or indirectly involved in the process of virus manufacture, including, but not limited to, viral gene expression, production of viral proteins, nucleic acids or other components, packaging of viral components into complete viruses, and cell lysis.
A "gene essential for replication" is a gene whose transcription is required for the viral vector to replicate in a cell.
As used herein, a "target cell" is a cell that allows (i.e., permits or induces) a cell type-specific transcriptional regulatory element (TRE) to function. Typically, the target cell is a mammalian cell, preferably a human cell.
As used herein, "cytotoxicity" is a term well understood in the art and refers to a state in which one or more of a cell's usual biochemical or biological functions are perturbed (i.e., inhibited or elevated). These activities include, but are not limited to, metabolism, cellular replication, DNA replication, transcription, translation, and uptake of molecules.
"Cytotoxicity" includes cell death and/or cytolysis. Assays are known in the art that indicate cytotoxicity, such as dye exclusion, 3H-thymidine uptake, and plaque assays.
The term "selective cytotoxicity", as used herein, refers to the cytotoxicity conferred by a viral vector of the present invention on a cell which allows a cell type-specific THE to function when compared to the cytotoxicity conferred by a viral vector of the invention on a cell which does not allow, or is less permissive for, the same THE to function. Such cytotoxicity may be measured, for example, by plaque assays, reduction or stabilization in size of a tumor comprising target cells, or the reduction or stabilization of serum levels of a marker characteristic of the tumor cells or a tissue-specific marker, e.g., a cancer marker such as prostate specific antigen.
As used herein, a "cytotoxic" gene is a gene whose expression in a cell, either alone or in conjunction with virus replication, enhances the degree and/or rate of cytotoxic and/or cytolytic activity in the cell.
A "therapeutic" gene is a gene whose expression in a cell is associated with a desirable result. In the cancer context, this desirable result may be, for example, cytotoxicity, repression or slowing of cell growth, and/or cell death.
A "biological sample" encompasses a variety of sample types obtained from an individual and can be used in a diagnostic or monitoring assay. The definition encompasses blood and other liquid samples of biological origin, solid tissue samples such as a biopsy specimen or tissue cultures or cells derived therefrom and the progeny. thereof.
The definition also includes samples that have been manipulated in any way after their procurement, such as by treatment with reagents, solubilization, or enrichment for certain components, such as proteins or polynucleotides. The term "biological sample"
encompasses a clinical sample, and also includes cells in culture, cell supernatants, cell lysates, serum, plasma, biological fluid, and tissue samples.

An "individual" is a vertebrate, preferably a mammal, more preferably a human.
Mammals include, but are not limited to, rodents, primates, farm animals, sport animals, and pets.
An "effective amount" is an amount sufficient to effect beneficial or desired clinical results. An effective amount can be administered in one or more administrations. For purposes of this invention, an effective amount of a viral vector is an amount that is sufficient to palliate, ameliorate, stabilize, reverse, slow or delay the progression of the disease state.
As used herein, "treatment" is an approach for obtaining beneficial or desired clinical results. For purposes of this invention, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, preventing spread (i.e., metastasis) of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable.
"Treatment" can also mean prolonging survival as compared to expected survival if not receiving treatment.
"Palliating" a disease means that the extent and/or undesirable clinical manifestations of a disease state are lessened and/or time course of the progression is slowed or lengthened, as compared to not administering viral vectors of the present invention.
Regulatable Transcrit~tional Transactivators and Repressors The present invention utilizes transactivating proteins that are functional in mammalian cells capable of serving as host cells for viral infection. Such transactivating proteins include synthetic, chimeric and naturally occurring transcriptional transactivating proteins or domains of proteins from eukaryotic cells including vertebrate cells, viral transactivating proteins or any synthetic amino acid sequence that is able to stimulate transcription from a vertebrate promoter. Such a transactivating protein may be (1 ) natural (native), (2) chimeric (chimera of a DNA-binding domain of a natural protein and a regulatory (activator or repressor) domain of a natural protein, (3) synthetic, having a novel DNA-binding domain designed by structural modeling, phage display screening or other methods, and (4) may or may not take the form of a fusion protein.
Types of transcriptional activation domains include acidic transcription activation domains, proline-rich transcription activation domains, serine/threonine-rich transcription activation domains and glutamine-rich transcription activation domains.
Examples of acidic transcriptional activation domains include the VP16 regions and amino acid residues 753-881 of Saccharomyces cerevisiae Gal4 (Braselmann et al., 1993, Proc Natl Acad Sci USA.
90 (5): 1657-1661 ). Examples of proline-rich activation domains include amino acid residues 399-499 of CTF/NF1 and amino acid residues 31-76 of AP2. Examples of serine/threonine-rich transcription activation domains include amino acid residues 1-427 of ITF1 and amino acid residues 2-451 of ITF2. Examples of glutamine-rich activation domains include amino acid residues 175-269 of Oct1 and amino acid residues 132-243 of Sp1. The amino acid sequences of each of the above described regions, and of other useful transcriptional activation domains, are disclosed in Seipel, K. et al. (EMBO
J. (1992) 13:4961-4968). Examples of transactivating proteins also include the lymphoid specific transcription factor identified by Muller et al. (1988, Nature 336:544-551), the fos protein (Lucibello et al., 1988, Oncogene 3:43-52); v-jun protein (Bos et al., 1988, Cell 52:705-712);
factor EF-C (Ostapchuk et al., 1989, Mol. Cell. Biol. 9:2787-2797); HIV-1 tat protein (Arya et al., 1985, Science 229:69-73), the papillomavirus E2 protein (Lambert et al., 1989, J. Virol.
63:3151-3154) and the adenovirus E1A protein (reviewed in Flint and Shenk, 1989, Ann.
Rev. Genet.). In preferred embodiments of the invention, the transactivating protein is Herpes simplex virus VP16 (Sadowski et al., 1988, Nature 335:563-564;
Triezenberg et al., 1988, Genes and Dev. 2:718-729). The human NF-kappaB activation domain (p65) has also been used in a number of systems (Vermeulen L et. al., Biochem Pharmacol 64(5-6):963-70, 2002).
Inducible transactivators include chimeric fusion proteins comprising (i) a functional portion of a DNA binding protein and (ii) a functional portion of a transcriptional activator protein, as described above. DNA sequences encoding the DNA binding protein and the transactivating protein are combined so as to preserve the respective binding and transactivating properties of each. Regions not required for function of DNA
binding proteins or transcriptional transactivating proteins may be identified by any method known in the art, including analysis of mapped mutations as well as identification of regions lacking mapped mutations, which are presumably less sensitive to mutation than other, more functionally relevant portions of the molecule. The appropriate recombinant constructs may be produced using standard techniques in molecular biology, including those set forth in Maniatis (1982, Molecular Cloning: A Laboratory Manual (Cold Spring Harbor, N.Y., Cold Spring Harbor Laboratory)).
The DNA binding protein portion may be derived from any vertebrate, nonvertebrate, fungal, plant, or bacterial source and may be natural, engineered or synthetic. Examples include Gal 4 (Keegan et al., 1986, Science 231:699-704), ADR1 (Hartshorne et al., 1986, Nature 320:283-287), Swl (Stillman et al., 1988, EMBO J. 7:485-495) and as generally reviewed in Johnson et al. (1989, Annual Rev. Biochem., 58:799-839). The transactivator may be a repressor protein, such as, for example, the IexA repressor. In some embodiments, a chimeric transactivator protein is derived from a bacterial DNA
binding protein, which then confers specificity on the transactivator protein by binding to sites engineered into the transcriptional transactivator regulated regulatory element. DNA
sequences homologous to bacterial DNA binding sites are unlikely to occur frequently in the mammalian genome, and therefore selectively control the expression of genes of interest.
Exemplary activator domains include but are not limited to VP16, NF-kappaB, TFE3, ITF1, Oct-1, Spl, Oct-2, NFY-A, ITF2, c-myc, and CTF (Seipel, et al., 1992, EMBO J
13: 4961-4968). Exempalry repressor domains include but are not limited to Kruppel (KRAB;
Margolin et al., 1994, Proc Natl Acad Sci USA 91 (10): 4509-13), kox-1 (Deuschle et al., 1995), even-skipped (Licht et al., 1994), LacR, engrailed (Li et al, 1997, J
Biol Chem., 274 (12): 7803-15), hairy (HES; Fisher et al., 1996, EMBO J 12 (13): 5075-82), Groucho (TLE;
Fisher et al., 1996), RING1 (Satjin et al., 1997, Mol. Cell. Biol. 17 (7):
4105-4113), SSB16 and SSB24 (Saha et al., 1993), Tupl (Tzamarlas, Struhl, 1994), Nabl (Swirnoff et al., 1998), AREB (Ikeda et al., 1998, Mol. Cell. Biol. 18 (1): 10-18), E4BP4 (Cowell ~
Hurst, 1996), HoxA7 (Schnabell et al, 1996), EBNA3 (Bourillot et al., 1998, J Gen Virol. 79 (Pt 2): 363-70.), and v-erbA (Busch et al., 1997, Mol Endocrinol. 11 (3): 379-89.). By way of specific example, the tTs (TET-silencer) uses the repression domain of the KRAB
protein. See also, PCT Publication WO0052179 for further examples of activator domains, repressor domains and chimeric transcriptional transactivators.
The chimeric transactivator protein may further comprise a nuclear localisation sequence, so that the chimeric transactivator protein is selectively concentrated in the cell nucleus. For example, nucleic acid sequence encoding the SV40 large T antigen nuclear localisation signal, Pro-Lys-Lys-Lys-Arg-Lys-Val (Kalderon et al., 1984, Cell 39:499-509) may be placed in apposition to the transactivator protein encoding sequences.
A number of methods for control of gene expression by inducing agents or conditions have been described, for example, those induced by tetracycline, antiprogestins, and ecdysone.
Inducible systems applicable to the current invention include the tet (TetT"~) and reverse (RevTetT"") systems, which are described in U.S. Pat. Nos. 5,464,758, 5,589,362, PCT publication WO 94/29442, PCT publication WO 96/01313 and PCT publication WO
96/40892, each of which is expressly incorporated by reference herein. For example, one may use the TetT"" and RevTetT"" systems (BD Biosciences Clontech), which employ small molecules, such as tetracycline (Tc) or analogues, e.g. doxycycline, to regulate (turn on or off) transcription of the target (Knott A et al., Biotechniques 32(4):796, 798, 800 (2002)).
Both the TetT"" and RevTetT"~ systems have been demonstrated to modulate gene expression in vivo. Modified vectors based on parvovirus, adenovirus, retroviruses and herpes simplex virus have been used to introduce the TET-Technology in vitro.
See, for example Ho, D.Y. et al. (1996) Mol. Brain Res. 41:200-209; Hwang, J-J. et al.
(1996) J.
Virol. 70:8138-814; Hu, S-X. et al. (1997) Cancer Res. 57:3339-3343; and Maxwell, I.H. et al. (1996) Gene Ther.3:28-36. Gossen et al. (1992) Proc Natl Acad Sci U S A.
89(12):5547-51 describe control of gene expression in mammalian cells by tetracycline-responsive promoters. Gossen et al. (1995) Science 268(5218):1766-9, describe transcriptional activation by tetracycline in mammalian cells. Urlinger et al. (2000) Proc Natl Acad Sci U S
A. 97(14):7963-8, discuss mutations in tetracycline-dependent transcriptional activators.
Freundlieb et al. (1999) J Gene Med. 1 (1 ):4-12 discuss tetracycline controlled activationlrepression system with increased potential for gene transfer into mammalian cells.
In another exemplary system, transcription is activated by rapamycin (or analogs thereof) which bring together two intracellular molecules, each of which is linked to either a transactivat~r or a DNA binding protein. When these components come together, transcription of the gene of interest is activated. Rapamycin mediates the formation of heterodimers between the immunophilin FK506-binding protein (FICBP) and the lipid kinase homolog FRAP (Standaert, R. F. et al., Nature 346, 671-674 (1990); Brown, E.
J. et al., Nature 369, 756-758 (1994); Rivers, V. M. et al. Nat. Med. 2, 1028-1032 (1996); and Ho, S.
N. et al., Nature 382, 822-826(1996)). Rapamycin-based systems (Ariad) have been shown to regulate target gene transcription both in cell culture and in animal models and high dose-dependent indu~cibility has been demonstrated following addition of rapamycin.
See, e.g., Ye et al. Science 283 (5398):88-91, 1999; Magari et al. J Clin Invest.
100(11):2865-72,1997;
Rivers et al. Nat Med. 2(9):1028-32,1996. Ariad has two major systems: a system based on homodimerization and a system based on heterodimerization (Rivers et x1.,1996, Nature Med, 2(9):1028-1032; Ye et al., 2000, Science 283: 88-91; Sawyer TK et al., 2002, Mini Rev Med Chem. 2(5):475-88). The heterodimerization system is based on human (FK506 binding protein) and a 93 as fragment of the large human P13F~ homolog, FRAP
(RAFT, mT~R). A DNA binding protein called ~.FHD1 (a fusion protein of two human DNA
binding domains) is joined to FKBP and the human NF-kappa b p65 activation domain is fused to FRAP (or if rapamycin analogs are used as inducers, instead a mutant version of FRAP). Upon addition of an inducer molecule, which binds to both FKBP12 and FRAP, FICBP12 and FRAP come together with the DNA binding protein and the activation domain.
Gene transcription is therefore initiated.
Other examples of regulated gene expression systems or promoters include the metallothionein promoter system (Mulherkar et al. Biochem Biophys Res Gommun.
177(1 ):90-6, 1991 ); glucocorticoid promoter systems (Ko et al. (1989) Gene 84(2):383-9), ecdysone-regulated gene switch (Saez et al. Proc Natl Acad Sci U S A.
97(26):14512-7) and the macrolide-based transgene control system (Weber et al. (2002) Nat Biotechnol.
20(9):901-7).

Among ecdysone systems are the Drosophila ecdysone system (Yao and Evans, 1996, Proc. Nat. Acad. Sci., 93:3346), the Bombyx ecdysone system (Suhr et al., 1998, Proc. Nat. Acad. Sci., 95:7999). Steroid based systems include a synthetic progesterone receptor system which employs RU-486 as the inducer (Wang et al., Biochim Biophys Acta, 1994, 1218 (3): 308-314; Delort and Capecchi, 1996, Hum Gene Ther 7 (7): 809-820; and ~sterwalder et al., 2001, Proc Natl Acad Sci 98(22):12596-601).
A "functionally-preserved" variant of a transcriptional transactivator, repressor or the response element therefor is a transcriptional transactivator, repressor or response element therefor which differs from a reference transcriptional transactivator, repressor or response element, but retains the ability to increase transcription of an operably linked polynucleotide, in particular, cell type-specific transcriptional activity. The difference can be due to an altered linear sequence or conformation, arising from, for example, single or multiple base mutation(s), addition(s), deletion(s), and/or modifications) of the bases. The difference can also arise from changes in the sugar(s), and/or linkages) between the bases.
The oncolytic viruses of the invention can be used for a wide variety of purposes.
The purpose will vary with the target cell. Suitable target cells are characterized by the transcriptional activation of the cell specific transcriptional response element in the viral vector. Regulation of transcriptional activation is the result of interaction between transcriptional activators bound to cis-regulatory elements, factors bound to basal ~transcriptional elements and the activity of transeriptional mediators, coactivafiors, and the presence of inducing agents or conditions. The transactivator regulated transcriptional regulatory element may be operably linked to a viral gene that is essential for propagation, so that replication competence is only achievable in the target cell, and/or to a transgene.
By transgene it is intended any gene that is not present in wild-type virus, frequently the transgene will also not be expressed in the target cell, prior to introduction by the virus.
One of skill in the art would recognize that some alterations of bases in and around transcription factor binding sites are more likely to negatively affect gene activation and cell-specificity, while alterations in bases which are not involved in transcription factor binding are not as likely to have such effects. Certain mutations are also capable of increasing transcriptional transactivator or repressor activity. Testing of the effects of altering bases may be performed in vitro or in viv~ by any method known in the art, such as mobility shift assays, or transfecting vectors containing these alterations in target non-target cells.
Additionally, one of skill in the art would recognize that point mutations and deletions can be made to a transcriptional transactivator or repressor sequence or the response element therefor without altering the ability of the sequence to regulate transcription.
It will be understood by one of skill in the art that very low basal levels of transcription may be present in non-targeted cell types. By transcriptional activation, it is intended that transcription will be increased above basal levels in the target cell by at least about 2-fold; preferably at least about 5-fold; preferably at least about 10-fold; more preferably at least about 20-fold, 30-fold or 40-fold; more preferably at least about 50-fold, 60-fold, 70-fold, i30-fol or 90-fold; more preferably at least about 100-fold;
even more preferably at least about 200-fold, even more preferably at least about 400-to about 500-fold, even more preferably, at least about 1000-fold.
All of the above-described systems for control of gene expression find utility in practicing the present invention. An effective dose of inducing agent or effective inducing conditions are readily determined from extrapolation of published results, from empirical testing, and other methods known and routinely employed by those of skill in the art. For example, the inducing agent may be administered to an animal harboring a viral vector of the present invention, and effective viral titers determined after administration of the inducing agent.
Transcri~tional Regulatory Elements (TREs) In practicing the current invention, the genetic sequence encoding the transactivator protein is desirably placed under the transcriptional control of a suitable cell type-specific TRE. A "cell type-specific TRE" is preferentially functional in a specific type of cell relative to other types of cells of difFerent functionality. "Cell type" is a reflection of a differenfiiation state of a cell which is, under normal physiological conditions, an irreversible, end-stage state. For example, a prostate-specific antigen THE is functional in prostate cells, but is not substantially functional in other cell types such as hepatocytes, astrocytes, cardiocytes, lymphocytes, etc. Generally, a cell type-specific THE is active in only one cell type. When a cell type-specific THE is active in more than one cell type, its activity is restricted to a limited number of cell types, i.e., it is not active in all cell types. A cell type-specific THE may or may not be tumor cell specific. Such cell type specificity refers to a relative increase in transcription in a target host cell wherein the THE is active relative to a cell wherein the THE
is not active.
As used herein, a THE derived from a specific gene is referred to by the gene from which it was derived and is a polynucleotide sequence which regulates transcription of an operably linked polynucleotide sequence in a host cell that expresses that gene.
Depending upon the target cell type, various enhancers may be used to provide for specific transcription. With lymphocytes, for B cells one may use the Ig enhancer, for T cells one may use the T cell antigen receptor promoter. For the different muscle cells, one may use the promoters for the different myosins. For endothelial cells, one may use the different promoters for the different selectins. For each type of cell, there will be specific proteins associated with the cell, which allows for target cell specific transcription.

As used herein, a "cell status-specific transcriptional regulatory element" or cell status specific THE is a THE that is induced or becomes active under a particular physiological state that permits or induces expression of polynucleotides under transcriptional control thereof.
An example of cell status is cell cycle. An exemplary gene whose expression is associated with the cell cycle is E2F-1, a ubiquitously expressed, growth-regulated gene, which exhibits peak transcriptional activity in S phase. Johnson et al. (1994) Genes Dev.
8:1514-1525. The RB protein, as well as other members of the RB family, form specific complexes with E2F-1, thereby inhibiting its ability to activate transcription. Thus, E2F-1-responsive promoters are down-regulated by RB. Many tumor cells have disrupted RB
function, which can lead to de-repression of E2F-1-responsive promoters, and, in turn, de-regulated cell division.
Included within the invention are replication competent vectors comprising a cell status-specific TRE, e.g., an "E2F-1-THE", a ubiquitously expressed, growth-regulated gene, which exhibits peak transcriptional activity in S phase. An "E2F-1-TRE"
is a polynucleotide sequence, preferably a ~NA sequence, which selectively increases transcription (of an operably-linked polynucleotide sequence) in a host cell that allows an E2F-1-THE to function, such as a cell (preferably a mammalian cell, even more preferably a human cell) that expresses E2F-1. The E2F-1-THE is responsive to transcription factors and/or co-factor(s) associated with E2F-1-producing cells and comprises at least a portion of the E2F-1 promoter and/or enhancer. See Hernandez-Alcoceba R et al. (Hum Gene Ther 2002 Sep 20;13(14):1737-50); Tsukuda et al. (Cancer Res. 62:3238-3447, 2002);
Johnson et al., Cancer Cell. 2002 May;1\(4):325-37. (~nyx); Jakubczak JL et al., Cancer Res. 2003 Apr 1;63(7):1490-9; United States Application Serial No. Serial No.
09/392,822, published as 20010053352 and United States Application Serial No. 10/081969, published as 20030104625. The sequence of the E2F promoter is known in the art, and has been described, e.g., in Fueyo J et al., Nat Med. 1998 Jun;4(6):685-90.
A further example of cell status is response to hypoxic conditions (such as a hypoxia response element or HRE). An important mediator of hypoxic responses is the transcriptional complex HIF-1, or hypoxia inducible factor-1, which interacts with a hypoxia-responsive element (HRE) in the regulatory regions of several genes, including vascular endothelial growth factor, and several genes encoding glycolytic enzymes, including enolase-1. Murine HRE sequences have been identified and characterized. Firth et al.
(1994) Proc. Natl. Acad. Sci. USA 91:6496-6500. Hypoxia is an integral component of the tumor microenvironment that develops in most solid tumors regardless of their origin, location, or genetic alterations and arises from the rapid growth of the tumor relative to its vascular supply. Since hypoxia is a major factor in conferring resistance of cancer cells to radiotherapy and chemotherapy, selecting tumor clones of high malignancy, predisposing tumors to metastasis, the development of novel therapeutic strategies that target hypoxic areas of tumors is important. Hypoxia-inducible factor (HIF) is a heterodimeric transcription factor that mediates responses to hypoxia by binding to a hypoxia-response element (HRE) present within target genes and the HIF/HRE system can therefore be utilized to specifically target therapeutic gene expression to tumors. Also included within the invention are replication competent vectors comprising a hypoxia responsive element or "HRE". A
hypoxia responsive element is a polynucleotide sequence, preferably a DNA
sequence, which selectively increases transcription (of an operably-linked polynucleotide sequence) in a host cell under conditions that allow a HRE to function, such as a cell (preferably a mammalian cell, even more preferably a human cell) that expresses hypoxia inducible factor-1, which interacts with a hypoxia-responsive element (HRE). The sequence of hypoxia-response elements are known in the art, e.g., an HRE from a rat enolase-1 promoter is described in Jiang et al. (1997) Cancer Res. 57:5328-5335.
Another exemplary gene whose expression is associated with the cell cycle is telomerase. See e.g., United States Application Serial No. 10/081969, published as 20030104625; PCT Publication WO00/46355; United States Application Serial No.

10/023,969, published as 20030095989 and United States Application Serial N~.
10/206447, published as US20030099616 (Geron); and United States Application Serial No. 09/956,335 published as 20020028785 (Saint Louis University), which describe regulated expression of adenovirus using the human telomerase promoter. The invention provides replication competent vectors comprising a telomerase regulatory element, e.g., a telomerase promoter. The sequences of a number of telomerase promoters are known in the ark, examples of which may be found in GenBank at Accession Nos.
AF128893.1 and AF121948.
Other cell status-specific transcriptional response elements include heat-inducible (i.e., heat shock) promoters, and promoters responsive to radiation exposure, including ionizing radiation and UV radiation. For example, the promoter region of the early growth response-1 (Egr-1) gene contains an elements) inducible by ionizing radiation.
Hallahan et al. (1995) Nat. Med. 1:786-791; and Tsai-Morris et al. (1988) Nucl. Acids.
Res. 16:8835-8846. Heat-inducible promoters, including heat-inducible elements, have been described.
See, for example Welsh (1990) in "Stress Proteins in Biology and Medicine", Morimoto, Tisseres, and Georgopoulos, eds. Cold Spring Harbor Laboratory Press; and Perisic et al.
(1989) Cell 59:797-806.
Accordingly, in some embodiments, a cell status-specific transcriptional response elements comprises one or more elements responsive to ionizing radiation, e.g., a 5' flanking sequence of an Egr-1 gene, a heat shock responsive, or heat-inducible element.

As used herein, the term "cell type-specific" is intended to mean that the THE
sequences to which a gene, i.e., a gene essential for viral replication, is operably linked, or to which a transgene is operably linked, functions specifically in that target cell so that transcription (and replication, if the operably linked gene is one essential for viral replication) proceeds selectively in target cells, or so that a transgene is expressed in target cells. This can occur by virtue of the presence in target cells, and not in non-target cells, of transcription factors that activate transcription driven by the operably linked transcriptional control sequences. It can also occur by virtue of the absence of transcription inhibiting factors that normally occur in non-target cells and prevent transcription driven by the operably linked transcriptional control sequences. The term "cell type-specific", as used herein, is intended to include cell type specificity, tissue specificity, as well as specificity for a cancerous state of a given target cell. In the latter case, specificity for a cancerous state of a normal cell is in comparison to a normal, non-cancerous counterpart.
In one embodiment, the invention includes oncolytic viral vectors wherein the CT-THE is prostate cell specific. For example, TREs that function preferentially in prostate cells and can be used in the present invention to target viral replication to prostate neoplasia, include, but are not limited to, TREs derived from the glandular kallikrein-1 gene (from the human gene, hf~CLdC2-TRE), the prostate-specific antigen gene (PSA-TRE), and the probasin gene (PB-TRE). All three of these genes are preferentially expressed in prostate cells and 2o the expression is androgen-inducible. Generally, expression of genes responsive to androgen induction requires the presence of an androgen receptor (AR).
Human glandular kallikrein (hd~CLdC2, encoding the hK2 protein) is expressed exclusively in the prostate and its expression is up-regulated by androgens primarily by transcriptional activation. The levels of hK2 found in various tumors and in the serum of patients with prostate cancer differ substantially from those of PSA and indicate that hK2 antigen may be a significant marker for prostate cancer.
A "human glandular kallikrein transcriptional regulatory element", or "hKLK2-TRE"
may be included in a replication competent vector of the invention. An "hKLK2-TRE" is a polynucleotide sequence, preferably a DNA sequence, which increases transcription of an operably linked polynucleotide sequence in a host cell that allows an hKLK2-THE to function, such as a cell (preferably a mammalian cell, even more preferably a human cell) that expresses androgen receptor. An hKLK2-THE is thus responsive to the binding of androgen receptor and comprises at least a portion of an hKLK2 promoter and/or an hKLK2 enhancer (i.e., the ARE or androgen receptor binding site). hKLK2-TREs are further described in US Application Serial No. 09/875,228.
The activity of the hKLK2 5' promoter has been previously described and a region up to -2256 relative to the transcription start site was previously disclosed (SEQ ID N0:3).

Schedlich et al. (1987) DNA 6:429-437. The hKLK2 promoter is androgen responsive and, in plasmid constructs wherein the promoter alone controls the expression of a reporter gene, expression of the reporter gene is increased approximately 10-fold in the presence of androgen. hKLK2 enhancer activity is found within a polynucleotide sequence approximately nt -12,014 to nt -2257 relative to the start of transcription (depicted in SEQ ID
N0:3) and, when this sequence is operably linked to an hKLK2 promoter and a reporter gene, transcription of operably-linked sequences in prostate cells increases in the presence of androgen at levels approximately 30- to approximately 100-fold over the level of transcription in the absence of androgen. This induction is generally orientation independent and position independent. Enhancer activity has also been demonstrated in the following regions (all relative to the transcription start site): about nt -3993 to about nt -3643 (nt 8021 to 8371 of SEQ ID N~:3), about nt -4814 to about nt -3643 (nt 7200 to 8371 of SEQ ID N~:3), about nt -5155 to about nt -3387 (nt 6859 to 8627 of SEQ ID
N~:3), about nt -6038 to about nt -2394 (nt 5976 to 9620 of SE(~ ID N~:3).
Thus, an hKLK2 enhancer can be operably linked to an hKLK2 promoter or a heterologous promoter to form an hKLK2 transcriptional regulatory element (hKLK2-TRE).
An hKLK2-THE can then be operably linked to a heterologous polynucleotide to confer hKLK2-TRE-specific transcriptional regulation on the linked gene, thus increasing its expression.
In another example, a "probasin (PB) transcriptional regulatory element", or "PB-TRE" is included in a replication competent vector of the invention. A or "PB-TRE" is a polynucleotide sequence, preferably a DNA sequence, which selectively increases transcription of an operably-linked polynucleotide sequence in a host cell that allows q PB-TRE to function, such as a cell (preferably a mammalian cell, even more preferably a human cell) that expresses androgen receptor. A PB-THE is thus responsive to the binding of androgen receptor and comprises at least a portion of a PB promoter and/or a PB
enhancer (i.e., the ARE or androgen receptor binding site). PB-TREs are further described in US Pat No. 6,436,394.
For example, the specificity of PB-THE activity for prostate cell that express the 3o androgen receptor (AR) was demonstrated as follows. The region of the PB 5'-flanking DNA (nt -426 to nt +28) (SEQ ID N~:9) including the endogenous promoter sequences was inserted upstream of the firefly luciferase gene to generate a chimeric PB-TRE-luc plasmid.
Cationic-mediated, transient transfection of LNCaP (PSA-producing and AR-producing prostate carcinoma cells) and PC-3 (PSA-deficient and AR-deficient prostate carcinoma cells) cells was performed. The results showed that LNCaP cells transfected with PB-TRE-luc had approximately 400 times more activity than untransfected cells, indicating that the PB-THE was intact. Further, the overall luciferase activity recovered in the cellular extracts of transfected LNCaP cells was about 30-40-fold higher than that measured in the cellular extracts of transfected PC-3 cells. Thus, the results indicate that PB-THE
expression is preferentially functional in PSA-producing, AR-producing prostate carcinoma cells as compared to PSA-deficient, AR-deficient prostate carcinoma cells and that PS-THE is capable of mediating specific expression in cells producing the androgen receptor. The rat probasin (PB) gene encodes a nuclear and secreted protein, probasin, that is only expressed in the dorsolateral prostate. A PB-THE has been shown in an approximately 0.5 kb fragment of sequence upstream of the probasin coding sequence, from about nt -426 to about nt +28 relative to the transcription start site, as depicted in (SEQ ID
NO:4). This minimal promoter sequence from the PB gene appears to provide sufficient informafiion to direct development and hormone -regulated expression of an operably linked heterologous gene specifically to the prostate in transgenic mice.
In another example, a "prostate-specific antigen (PSA) transcriptional regulatory element", or "PSA-THE", or "PSE-TRE" is included in a replication competent vector of the invention. A "PSA-THE", or "PSE-TRE" is a polynucleotide sequence, preferably a DNA
sequence, which selectively increases transcription of an operably linked polynucleotide sequence in a host cell that allows a PSA-THE to function, such as a cell (preferably a mammalian cell, even more preferably a human cell) that expresses androgen receptor. A
PSE-THE is thus responsive to the binding of androgen receptor and comprises at least a portion of a PSA promoter and/or a PSA enhancer (i.e., the ARE or androgen receptor binding site). PSE-TREs are further described in US Patent Nos. 5,648,478, 6,057,299 and 6,136, 792.
The region of the PSA gene that is used to provide cell specificity dependent upon androgens, particular in prostate cells, involves approximately 6.0 kilobases.
Schuur et al.
(1996) J. viol. Chew. 271:7043-7051. An enhancer region of approximately 1.5 kb in humans is located between nt -5322 and nt -3739, relative to the transcription start site of the PSA gene. The PSA promoter consists of the sequence from about nt -540 to nt +8 relative to the transcription start site. Juxtapositioning of these two genetic elements yields a fully functional, minimal prostate-specific enhancer/promoter (PSE) TRE.
Other portions of the approximately 6.0 kb region of the PSA gene can be used in the present invention, as long as requisite functionality is maintained.
The PSE and PSA THE depicted in (SEQ ID NO:1) is the same as that given in GenBank Accession No. U37672. A variant PSA-THE nucleotide sequence is depicted in (SEQ ID N0:2). This is the PSA-THE contained within CN706 clone 35.190.13.
CN706 is an adenoviral vector in which the E1A gene in Ad5 is under transcriptional control of a PSA-TRE. CN706 demonstrates selective cytotoxicity toward PSA-expressing cells in vitro and in vivo. Rodriguez et al. (1997).

The region that is employed to provide cell specificity dependent upon androgens, particularly in prostate cells, involves an approximately 1.5kb enhancer region and a 0.5kb promoter region. The enhancer region in humans is located between nt -5322 and nt -3739, relative to the transcription start site of the prostate specific antigen (PSA) gene. The promoter consists of nt -540 to nt +8. Juxtaposition of the two genetic elements yields a fully functional, minimal prostate-specific enhancer promoter (PSE). The enhancer contains three regions that bind prostate-specific DNA binding proteins, one of which contains a putative androgen response element. The promoter region contains typical TATA
and CHAT boxes as well as a second putative androgen response element.
CEA is a 180,000-Dalton glycoprotein tumor-associated antigen present on endodermally-derived neoplasia of the gastrointestinal tract, such as colorectal, gastric (stomach) and pancreatic cancer, as well as other adenocarcinomas such as breast and lung cancers. In yet another example, a "carcinoembryonic antigen (CEA) transcriptional regulatory element", or "CEA-TRE" is included in a replication competent vector of the invention. A "CEA-THE is a polynucleotide sequence, preferably a DNA sequence, which selectively increases transcription of an operably linked polynucleotide sequence in a host cell that allows a CEA-THE to function, such as a cell (preferably a mammalian cell, even more preferably a human cell) that expresses CEA. The CEA-THE is responsive to transcription factors and/or co-factor(s) associated with CEA-producing cells and comprises at least a portion of the CEA promoter and/or enhancer. The 5' upstream flanking sequence of the CEA gene has been shown to confer cell-specific activity. The CEA
promoter region, approximately the first 424 nucleotides upstream of the translational start site in the 5' flanking region of the gene, was shown to confer cell-specific activity when the region provided higher promoter activity in CEA-producing cells than in non-producing HeLa cells. In addition, cell-specific enhancer regions have been found. The entire 5' CEA
flanking region (containing the promoter, putative silencer, and enhancer elements) appears to be contained within approximately 14.5 kb upstream from the transcription start site. Two upstream regions, -13.6 to -10.7 kb or -6.1 to -4.0 kb, when linked to the multimerized promoter resulted in high-level and selective expression of a reporter construct in CEA-producing cells. The promoter region is localized to nt -90 and nt +69 relative to the transcriptional start site, with region nt -41 to nt -18 as essential for expression.
W095/14100 describes a series of 5' flanking CEA fragments which confer cell-specific activity, such as about nt -299 to about nt +69; about nt -90 to about nt +69;
nt -14,500 to nt -10,600; nt -13,600 to nt -10,600, nt -6100 to nt -3800. In addition, cell specific transcription activity is conferred on an operably linked gene by the CEA fragment from nt -402 to nt +69, depicted in (SEQ ID N0:7). CEA-TREs used in the present invention are derived from mammalian cells, including but not limited to, human cells. Thus, any of the CEA-TREs may be used in the invention as long as requisite desired functionality is displayed. The cloning and characterization of CEA sequences have been described in the literature (e.g., in US Application Serial No. 10/045,116, published as 200030026792).
In a further example, an "alpha-fetoprotein (AFP) transcriptional regulatory element", or "AFP-TRE" may be included in a replication competent vector of the invention. AFP is an oncofetal protein. The serum concentration of AFP is elevated in a majority of hepatoma patients, with high levels of AFP found in patients with advanced disease. The serum AFP
levels in patients appear to be regulated by AFP expression in hepatocellular carcinoma buff not in surrounding normal liver. Thus, the AFP gene appears associated with hepatoma cell-specific expression. Gell-specific TREs from the AFP gene have been identified. An "AFP-TRE" is a polynucleotide sequence, preferably a DNA sequence, which selectively increases transcription (of an operably linked polynucleotide sequence) in a host cell that allows an AFP-THE to function, such as a cell (preferably a mammalian cell, even more preferably a human cell) that expresses AFP. The AFP-THE is responsive to transcription factors and/or co-factor(s) associated with AFP-producing cells and comprises at least a portion of the AFP promoter and/or enhancer. AFP-TREs are further described in US
Application Serial No. 09/898,883, expressly incorporated by reference herein.
The entire 5' AFP flanking region (containing the promoter, putative silencer, and enhancer elements) is contained within approximately 5 kb upstream from the transcription start site (SEQ ID
N~:5).
The AFP enhancer region in humans is located between about nt -3954 and about nt -3335, relative to the transcription start site of the AFP gene. The human AFP promoter encompasses a region from about nt -174 to about nt +29. Juxtapositioning of these two genetic elements, yields a fully functional AFP-TRE. Ido et al. (1995) describe a 259 by promoter fragment (nt -230 to nt +29) that is specific for HCC. Censer Res.
55:3105-3109.
The AFP enhancer contains two regions, denoted A and B, located between nt -3954 and nt -3335 relative to the transcription start site. The promoter region contains typical TATA and CART boxes. Preferably, the AFP-THE contains at least one enhancer region.
More preferably, the AFP THE contains both enhancer regions.
Suitable target cells for oncolytic viral vectors containing AFP-TREs are any cell type that allows an AFP-THE to function. Preferred are cells that express, or produce, AFP, including, but not limited to, tumor cells expressing AFP. Examples of such cells are hepatocellular carcinoma cells, gonadal and other germ cell tumors (especially endodermal sinus tumors), brain tumor cells, ovarian tumor cells, acinar cell carcinoma of the pancreas, primary gall bladder tumor, uterine endometrial adenocarcinoma cells, and any metastases of the foregoing (which can occur in lung, adrenal gland, bone marrow, and/or spleen). In some cases, metastatic disease to the liver from certain pancreatic and stomach cancers produces AFP. Especially preferred are hepatocellular carcinoma cells and any of their metastases. AFP production can be measured using assays standard in the art, such as RIA, ELISA or Western blots (immunoassays) to determine levels of AFP protein production or Northern blots to determine levels of AFP mRNA production. Alternatively, such cells can be identified and/or characterized by their ability to activate transcriptionally an AFP-THE
(i.e., allow an AFP-THE to function).
In yet a further example, a urothelial cell-specific transcriptional regulatory element is included in a replication competent vector of the invention. A urothelial cell-specific transcriptional regulatory element is a polynucleotide sequence, preferably a DNA
sequence which selectively increases transcription (of an operably-linked polynucleotide sequence) in a urothelial host cell. For example, a urothelial cell-specific THE may be derived from the 5' flanking region of a uroplakin gene (i.e., a "UP-THE") such that the THE
selectively increases transcription (of an operably-linked polynucleotide sequence) in a cell that allows a UP-THE to function such as a cell (preferably a mammalian cell, even more preferably a human cell) that expresses uroplakin. In some of these embodiments, the urothelial cell-specific THE is derived from the 5' flanking region of a UPIa gene. In other embodiments, the urothelial cell-specific THE is derived from the 5'-flanking region of a UPIb gene. In yet other embodiments, the urothelial cell-specific THE is derived from the 5'-flanking region of a UPII gene. The UP-THE may comprise a urothelial cell-specific promoter and a heterologous enhancer. In other embodiments, a urothelial cell-specific THE
comprises a urothelial cell-specific promoter. In other embodiments, a urothelial cell-specific THE comprises a urothelial cell-specific enhancer and a heterologous promoter. In other embodiments, a urothelial sell-specific THE comprises a urothelial cell-specific promoter and a urothelial cell-specific enhancer. UP-TREs are further described in PCT
publication WO 01/72994..
The protein product of the MUC1 gene (known as mucin or MUC1 protein;
episialin;
polymorphic epithelial mucin or PEM; EMA; DF3 antigen; NPGP; PAS-O; or CA15.3 antigen) is normally expressed mainly at the apical surface of epithelial cells lining the glands or ducts of the stomach, pancreas, lungs, trachea, kidney, uterus, salivary glands, and mammary glands. However, mucin is overexpressed in 75-90% of human breast carcinomas. Mucin protein expression correlates with the degree of breast tumor differentiation. This overexpression appears to be controlled at the transcriptional level.
Overexpression of the MUC1 gene in human breast carcinoma cells MCF-7 and ZR-appears to be regulated at the transcriptional level. A "MUC1-TRE" is a polynucleotide sequence, preferably a DNA sequence, which selectively increases transcription (of an operably-linked polynucleotide sequence) in a host cell that allows an MUC1-THE to function, such as a cell (preferably a mammalian cell, even more preferably a human cell) that expresses MUC1. The MUC1-THE is responsive to transcription factors and/or co-factors) associated with MUC1-producing cells and comprises at least a portion of the MUC1 promoter and/or enhancer. MUC1-TREs are further described in US Patent No.
6,432,700. In an additional example, "a mucin gene (MUC) transcriptional regulatory element", or "MUC1-TRE" is included in a replication competent vector of the invention.
The regulatory sequences of the MUC1 gene have been cloned, including the approximately 0.9 kb upstream of the transcription start site which contains a THE that appears to be involved in cell-specific transcription, depicted in SEQ ID
NO:B.
Any MUC1-TREs used in the present invention are derived from mammalian cells, including but not limited to, human cells. Preferably, the MUC1-THE is human.
In one embodiment, the MUC1-THE may contain the entire 0.9 kb 5' flanking sequence of the MUC1 gene. In other embodiments, the MUC1-TREs comprise the following sequences (relative to the transcription start site of fihe MUC1 gene): about nt -725 to about nt +31, nt -743 to about nt +33, nt -750 to about nt +33, and nt -598 to about nt +485 (operably-linked to a promoter).
The c-erbB2/neu gene (HER-2/neu or HER) is a transforming gene that encodes a 185 kD epidermal growth factor receptor-related transmembrane glycoprotein. In humans, the c-erbB2/neu protein is expressed during fetal development, however, in adults, the protein is weakly detectable (by immunohistochemistry) in the epithelium of many normal tissues. Amplification and/or over-expression of the c-erb~2/neu gene has been associated with many human cancers, including breast, ovarian, uterine, prostate, stomach and lung cancers.
In yet an additional example, "a HER-2/neu transcriptional regulatory element", or "HER-2/neu -TRE" is included in a replication competent vector of the invention.
Additional tumor- and/or cell type-specific TREs known in the art which may be included in the oncolytic vectors of the invention include the following:
aromatase, mammary gland-specific promoter, mammaglobin, plasminogen activator urokinase (uPA) and its receptor gene (associated with breast, colon, and liver cancers; TREs that regulate uPA and uPAR transcription are provided in SEQ ID N0:6 and further described in Riccio et al. Nucleic Acids Res. 13:2759-2771, 1985; Cannio et al., Nucleic Acids Res. 19:2303-2308, 1991); human alpha-lactalbumin (associated with breast tissue); BCSG1, BRCA1, and BRCA2 (associated with breast cancer); human papilloma virus (HPV) cell type dependent regulatory elements (associated with cervical cancer); BLCA4 (associated with bladder cancer); uroplakins (associated with bladder); NCA (associated with gastric cancer);
hypoxanthine phosphoribosyltransferase (HPRT; associated with glioma); AVP, human pulmonary surfactant protein B gene, and puromycin N-acetyltransferase associated with (associated with lung cancer); tyrosinase, gp100; melanoma specific TREs, such as the human MART promoter (hMART), the murine tyrosinase gene enhancer and promoter (mEP), a THE comprising a murine tyrosinase gene enhancer and promoter (mEEP), a THE
comprising the human tyrosinase promoter (hTYR) and melanocyte specific factory (MSF);
HER2/neu, urokinase, and CA125 (associated with ovarian cancer); SL3-3 and T
cell antigen receptor (associated with T cell lymphoma); prostatic acid phosphatase (associated with prostate cancer); an EBV-specific THE (associated with EBV-expressing tumors) such as an LMP1 Promoter; an LMP2A Promoter, an LMP2B Promoter; or a Cp Promoter (as descried e.g., in Sjoblom, A et al, J.Virol. (1998) 72, (2), 1365-1376;
Franken et al, J. Virol (1995) 69 (12) 8011-8019.; Laux et al, J. Gen. Virol, 1989, 70, 3079-3084);
and Fuentes-Panana et al, J. Virol., (1999) 73, 826-833 and V01555); a liver specific THE
(associated with liver cancer) such as a CRGL2 promoter; and a WT1 promoter and enhancer for leukemia specific gene expression (Hosen N. Leukemia. 2004 Jan 22). In addition, tumor specific promoters may be derived by chimeric construction using different promoter elements as exemplified by the artificial hTERT-cHSF1/HSE promoter (described for example in Wang J et al. FEBS Lett. Jul 10;546 (2-3):315-20, 2003 or completely synthetic in construction (Li X et al Nat Biotechnol. 1999 Mar;17(3):241-5).
Descriptions for these cell-specific TREs can be found in various scientific publications, and numerous promoter, enhancer and regulatory sequences associated with these TREs may be found in the GenBank database available on the Internet at htt~://www.ncbi.nlm.nih.aovlPubMed/. Many relevant sequences are thus available for practice of this invention and need not be described in detail herein.
The TREs listed above are provided as non-limiting examples of TREs that function in the instant invention. Additional cell-specific TREs are known in the art, as are methods to identify and test cell specificity of candidate TREs. A THE may or may not lack a silencer.
The presence of a silencer (i.e., a negative regulatory element) may assist in shutting off transcription (and thus replication) in non-permissive cells (i.e., cells in a normal cell state).
Thus, the presence of a silencer may confer enhanced inducible or cell-specific replication by more effectively preventing oncolytic viral vector replication in non-target cells.
Alternatively, the lack of a silencer may assist in effecting replication in target cells, thus conferring enhanced cell type-specific replication due to more effective replication in target cells. A THE can also comprise multimers. For example, a THE can comprise a tandem series of at least two, at least three, at least four, or at least five TREs.
These multimers may also contain heterologous promoter and/or enhancer sequences.
In one exemplary embodiment, two substantially identical TREs control transcription of viral genes, e.g. adenoviral E1A and E1 B genes. It is understood, however, that any of a number of combinations of genes may be used with these at least two TREs. In the adenoviral example, other preferred embodiments include those which contain substantially identical TREs that drive expression of E1A, E1 B, and E4. Such constructs may or may not additionally contain a transgene, which may or may not be under control of a substantially identical TRE. Preparation of these and other embodiments are provided below and in the examples.
Transcriptional activation can be measured in a number of ways known in the art (and described in more detail below), but is generally measured by detection and/or quantitation of mRNA or the protein product of the coding sequence under control of (i.e., operatively linked to) the TRE. Activity of a THE can be determined as follows. A THE
polynucleotide sequence or set of such sequences can be generated using methods known in the art, such as chemical synthesis, site-directed mutagenesis, PCR, and/or recombinant methods. The sequences) to be tested can be inserted into a vector containing a promoter (if no promoter element is present in the TRE) and an appropriate reporter gene encoding a reporter protein, including, but not limited to, chloramphenicol acetyl transferase (CAT), beta-galactosidase (encoded by the lacZ gene), luciferase (encoded by the luc gene), alkaline phosphatase, green fluorescent protein, and horseradish peroxidase.
Such vectors and assays are readily available, from, inter alia, commercial sources.
Plasmids thus constructed are transfected into a suitable host cell to test for expression of the reporter gene as controlled by the putative THE using transfection methods known in the art, such as calcium phosphate precipitation, electroporation, liposomes (lipofection), and DEAE
dextran.
After introduction of the TRE-reporter gene construct into a host cell under appropriate conditions, THE activity may be measured by detection and/or quantitation of reporter gene-derived mRNA or protein product(s), including in the absence of presence of a suitable inducing agent or condition. The reporter gene protein can be detected directly (e.g., immunochemically) or through its enzymatic activity, if any, with an appropriate substrate. Generally, to determine cell specific activity of a TRE, the TRE-reporter gene constructs are introduced into a variety of cell types. The amount of THE
activity is determined in each cell type and compared to that of a reporter gene construct without the TRE. A THE is cell specific when it is preferentially functional in a specific type of cell over a different type of cell.
As used herein, "a cell that allows a THE to function" or a cell in which the function of a THE is "sufficiently preserved" or "functionally preserved", or "a cell in which a THE is functional" is a cell in which the TRE, when operably linked to a promoter (if not included in the TRE) will increase transcription above basal levels in the target cell by at least about 2-fold, preferably at least about 5-fold, preferably at least about 10-fold, more preferably at least about 20-fold, more preferably at least about 50-fold, more preferably at least about 100-fold, even more preferably at least about 200-fold, even more preferably at least about 400- to about 500-fold, even more preferably, at least about 1000-fold. Basal levels are generally the level of activity, if any, in a non-target cells, or the level of activity (if any) of a reporter construct lacking the THE of interest as tested in a target cell type. Methods for measuring levels of expression (whether relative or absolute) are known in the art and are described herein. Similarly, when employing the regulatable oncolytic virus.systems of the invention, an inducible transactivator in the presence of an effective amount of inducing agent or under inducing conditions sufficient to induce gene expression, will increase expression of a gene at least about 2-fold, preferably at least about 5-fold, preferably at least about 10-fold, more preferably at least about 20-fold, more preferably at least about 50-fold, more preferably at least about 100-fold, more preferably at least about 200-fold, even more preferably at least about 400- to about 500 fold, even more preferably at least about 1000-fold, when compared to the expression of the same promoter and gene in the absence of the inducing agent.
A "functionally-preserved" variant of a THE is a THE which differs from anofiher TRE, but still retains the ability to increase transcription of an operably linked polynucleotide, in particular, cell type-specific transcription activity. The difference in a THE
can be due to differences in linear sequence or conformation, arising from, for example, single or multiple base mutation(s), addition(s), deletion(s), and/or modification(s). The difference can also arise from changes in the sugar(s), and/or linkages) between the bases of a TRE.
Certain point mutations within sequences of TREs have been shown to decrease transcription factor binding and gene activation. One of skill in the art would recognize that some alterations of bases in and around known fihe transcription factor binding sites are more likely to negatively affect gene activation and cell-specifiicity, while alterations in bases which are not involved in transcription factor binding are not as likely to have such effects.
Certain mutations are also capable of increasing THE activity. Testing of the effects of altering bases may be performed in vitro or in vivo by any method known in the art, such as mobility shift assays, or transfecting vectors containing these alterations in THE functional and THE non-functional cells. Additionally, one of skill in the art would recognize that point mutations and deletions can be made to a THE sequence without altering the ability of the sequence to regulate transcription.
_System For External Control Of Oncolytic Virus Replication Replication-competent (oncolytic) viral vectors are provided. The vectors comprise a cell type-specific transcriptional regulatory element (CT-TRE) and an inducible transactivator regulated transcriptional regulatory element. The CT-THE
controls transcription of an inducible transactivator (TA) coding sequence. The inducible transactivator (a) requires an inducing agent or condition to be functional and (b) controls transcription of a viral gene essential for replication. Expression of the viral gene essential for replication is regulated both by the CT-THE and by a transactivator regulated transcriptional regulatory element, and indirectly by the concentration of the inducing agent or condition.
Depending on the specific uses of the virus vector, the inducing agent or condition may act to inhibit transcription, or to enhance transcription. Transcription inhibitors provide a way to "shut down" viral replication by delivering the inducing agent to the host cells.
Transcription activators provide a way to induce virus replication by addition of the inducing agent, where the virus is otherwise inactive.
Included within the scope of the invention is any oncolytic virus wherein control of a viral gene essential for replication may be accomplished using the methods of the present invention. Exemplary replication-competent (oncolytic) viruses include, but are not limited to, adenoviruses, vesicular stomatitis viruses (VSV), herpes viruses (e.g., herpes simplex virus; HSV), reoviruses, paramyxoviruses, rhinoviruses, Newcastle disease viruses, polioviruses, West Nile virus, coxsaclcie virus, measles viruses and vaccinia viruses, etc.
In embodiments in which two substantially identical TREs are used, the invention does not require that the TREs be derived from the same gene. As long as the THE
sequences are substantially identical, and the requisite functionality is displayed, the TREs may be derived from different genes.
In some embodiments, the oncolytic vectors of the invention comprise a first viral gene under the transcriptional control of a transactivator regulated transcriptional regulatory element and at least one other gene, such as a viral gene or a transgene, under control of another heterologous THE which is different from the first TRE, where the heterologous TREs are functional in the same cell but do not have the same in polynucleofiide sequence (i.e., have different polynucleotide sequences). Preferably, at least two of the heterologous TREs in the oncolytic vector are cell specific or inducible for the same cell or inducing agent or condition. In one approach, the viral gene is one that enhances cell death, more preferably one that is essential for viral replication. Preferably, at least one of the viral genes necessary for cell replication is an early gene and the genes under transcriptional control of the heterologous THE are necessary for replication. By providing for cell-specific transcription through the use of multiple heterologous TREs, the invention provides oncolytic vectors that can effect cell-specific cytotoxic effects due to selective replication.
The novel system of the invention for regulated expression of oncolytic viruses is exemplified herein by regulatable adenoviral vectors. In this exemplary embodiment, the genes that are regulated by the transactivator regulated transcriptional regulatory element may be early or late adenoviral genes and/or transgenes. By providing for regulated transcription, one can provide for adenovirus that can be used as a vehicle for introducing genetic capability into host target cells, as distinct from other non-target cell types. The transgenes serve to modify the genotype or phenotype of the target cell, in addition to any modification of the genotype or phenotype resulting from the presence of the adenovirus.
Typical of the oncolytic vectors of the invention, with replication competent adenoviruses, proliferation of the adenovirus may be used for its cytotoxic effect.
It has been demonstrated that adenovirus vectors which include at least two different heterologous TREs are more stable and provide greater cell specificity with regard to replication than previously described adenovirus vectors. See, e.g., U.S.
Patent No.
6,4.32,700. Accordingly, adenovirus vectors have been constructed in which each of the E1A and E1 B genes are under transcriptional control of two different heterologous TREs. It is understood, however, that any of a number of combinations of genes may be used with any combinati~n of at least two TREs.
There are a number of different serotypes of adenovirus, such as Ad2, AdS, Ad3, Ad35 and Ad40, which differ to minor or significant degrees. Particularly, adenoviral serotypes differ as to host cell tropism. For the purpose of the subject invention, Ad5 is exemplified, however any and all serotypes of adenovirus are included within the scope of the invention.
The genes of the adenovirus that are of interest for the subject invention may be divided into two groups, the early genes and the late genes, the expression of the latter being controlled by the major late promoter. Of the early genes, there are E1A, E1 B, E2, E3 and E4.. The E1A gene is expressed immediately after viral infection (0-2h) and before any other viral genes. E1A protein acts as a trans-acting positive-acting transcriptional regulatory factor, and is required for the expression of the other early viral genes and the promoter proximal major late genes. Despite the nomenclature, the promoter proximal genes driven by the major late promoter are expressed during early times after Ad5 infection. In the absence of a functional E1A gene, viral infection does not proceed, because the gene products necessary for viral DNA replication are not produced.
The E1 B protein functions in trans and is necessary for transport of late mRNA from the nucleus to the cytoplasm. Defects in E1 B expression result in poor expression of late viral proteins and an inability to shut off host cell protein synthesis.
The E4 gene has a number of transcription products. Open reading frames (ORF) and ORF 6 of the E4 transcription unit increase the accumulation of major late transcription unit mRNAs by binding the 55-kDa protein from E1 B and heterodimers of E2F-1 and DP-1.
In the absence of functional protein from ORF3 and ORF6, plaques are produced with an efficiency less than 106 of that of wild type virus.
The major late genes relevant to the subject invention are genes such as LI, L2 and L3, which encode proteins of the AD5 virus virion.

Regions of the adenovirus which may be deleted, usually at least 500 nt, more usually at least about 1000 nt, include in the AD5 genome nucleotides 300 to 3600 in EI, particularly 342 to 3523; 27000 to 31000, particularly 28133 to 30818 or 27865 to 30995 in E3. The deletion will be at least sufficient for insertion of the desired construct and allow for packaging. In some embodiments E3 sequences are included in the regulatable replication competent adenoviruses of the invention, as further described in US
Application Patent No.
6,495,130. In other embodiments, internal ribosome entry sites (IRES) are included in the regulatable replication competent adenoviruses of the invention, as further described in US
Application Serial No. 09/814,351, published as 20030148520.
As exemplified by employing an adenoviral vector with a cell specific response element comprising a promoter and enhancer construct specific for prostate cells, various genetic capabilities may be introduced into prostate cells expressing prostate specific antigen. ~f particular interest is the opportunity to introduce cytotoxic effects that are controlled by a transcriptional initiation region specifically active in prostate cells. ~ther cell types that have specific active transcription factors associated with a state for which modulation is desirable include leukocytes, particularly lymphocytes, epithelial cells, endothelial cells, hepatic cells, pancreatic cells, neuronal cells, and keratinocytes. Since the adenovirus typically results in transient expression (approximately 6 to 8 weeks), one can provide transient capability to cells, for example in situations where the desired result only requires a limited period for response. In other cases, a different oncolytic vector may be preferred due the time and level of expression desired.
To further increase specificity of the control mediated by an inducing agent, it may also be desirable to control expression of 2 viral genes, i.e. expression of both E1A and E1 B adenoviral genes. In this manner, specificity may be increased, i.e., replication of the oncolytic virus in non-target cells in which the CT-THE is not active may be reduced further.
Both E1A and E1 B may be controlled by the inducer responsive promoter element as exemplified in Figure 1, which illustrates a prostate specific oncolytic viral vector with tetracycline regulated replication control, wherein a tetracycline responsive element (TRE) promoter drives E1A gene expression and a prostate specific Antigen (PSA) promoter drives expression of the reverse tet-responsive transactivator (rtTA).
In this approach expression of the E1A and E1B genes may be linked by an IRES
between the E1A and E1 B genes. In the construction of this virus, the endogenous E1 B
promoter elements are removed and replaced with the IRES element. Therefore both E1A
and E1 B expression are under the control of the inducer responsive promoter element. As an IRES alternative, the 2A peptide sequence derived foot and mouth disease virus (FMDV) could be used in place of the IRES sequence (as described in Furler S et al., Gene Ther.

2001 Jun;B(11):864-73) to provide efficient bicistronic expression of both E1A
and a transgene.
Preferred adenoviral embodiments include those that contain at least two different heterologous TREs that drive expression of E1A, E1B, and E4. Such constructs may or may not additionally contain a transgene, which may or may not be under control of a TRE, wherein the THE may or may not be a CT-TRE. See, e.g., US Patent Nos.
6,436,394 and 6,432,700.
Accordingly, the invention provides an oncolytic virus vector comprising a viral gene under transcriptional control of a transactivator regulated transcriptional regulatory element, and an inducible transactivator under transcriptional control of a TRE, preferably a CT-TRE.
In some embodiments, a first transactivator regulated transcriptional regulatory element controls expression of a first viral gene, and a second transactivator regulated transcriptional regulatory element controls expression of a second viral gene.
The genes to be controlled under these TREs are preferably viral genes essential for propagation.
Alternatively, the genes to be controlled under these TREs may be a first viral gene essential for propagation wherein the second gene is a transgene. It is understood that there may or may not be additional TREs in the viral vectors, and that these additional TREs may or may not be substantially identical to the first and/or second TREs. The invention includes use of three or more, four or more, TREs.
Transaenes Use of viral vectors, competent in particular target cells, allows for proliferation of the virus in the target cells resulting in the death of the host cells and proliferation of the virus to other host cells. To further enhance therapeutic efficacy, the oncolytic vectors of the invention may include one or more transgenes that have a therapeutic effect.
Accordingly, the viral vectors of this invention can further include a heterologous polynucleotide (transgene) encoding a therapeutic gene product under the control of a transactivator regulated transcriptional regulatory element. Alternatively, the viral vector may comprise a heterologous transgene encoding a therapeutic gene product under the control of a constitutive or inducible promoter. Numerous examples of constitutive and inducible promoters are known in the art and routinely employed in transgene expression in the context of viral vectors. In this way, various genetic capabilities may be introduced into target cells. For example, in certain instances, it may be desirable to enhance the degree therapeutic efficacy by enhancing the rate of cytotoxic activity. This could be accomplished by coupling the cell-specific replicative cytotoxic activity with expression of, one or more metabolic enzymes such as HSV-tk, nitroreductase, cytochrome P450 or cytosine deaminase (cd) which render cells capable of metabolizing 5-fluorocytosine (5-FC) to the chemotherapeutic agent 5-fluorouracil (5-FU) and carboxypeptidase G2 (CPG2).
This type of transgene may also be used to confer a bystander effect.
Additional transgenes that may be introduced into a viral vector of the invention include a factor capable of initiating apoptosis, antisense or ribozymes, which among other capabilities may be directed to mRNAs encoding proteins essential for proliferation, such as structural proteins, transcription factors, polymerases, etc., viral or other pathogenic proteins, where the pathogen proliferates intracellularly, cytotoxic proteins, e.g., the chains of diphtheria, ricin, abrin, etc., genes that encode an engineered cytoplasmic variant of a nuclease (e.g., RNase A) or protease (e.g., trypsin, pepsin, proteinase K, carboxypeptidase, 1o etc.), chemokines, such as MCP3 alpha or MIP-1, pore-forming proteins derived from viruses, bacteria, or mammalian cells, fusgenic genes, chemotherapy sensitizing genes and radiation sensitizing genes. ~ther genes of interest include cytokines, antigens, transmembrane proteins, and the like, such as IL-1, -2, -6, -12, GM-CSF, G-CSF, M-CSF, IFN-a, -~3, -y, TNF-a, -Vii, TGF-a, -Vii, NGF, MDA-7 (Melanoma differentiation associated gene-7, mda-7/interleukin-24), and the like. Further examples include, proapoptotic genes such as Fas, Bax, Caspase, TRAIL, Fas ligands, and the like; fusion genes which can lead to cell fusion or facilitate cell fusion such as V22, VSV and the like; tumor suppressor gene such as p53, RB, p16, p17, W9 and the like; genes associated with the cell cycle and genes which encode anti-angiogenic proteins such as endostatin, angiostatin and the like.
~ther opportunities for specific genetic modification include T cells, such as tumor infiltrating lymphocytes (TILs), where the TILs may be modified to enhance expansion, enhance cytotoxicity, reduce response to proliferation inhibitors, enhance expression of lymphokines, etc. ~ne may also wish to enhance target cell vulnerability by providing for expression of specific surface membrane proteins, e.g., B7, SV4.0 T antigen mutants, etc.
In some embodiments, the adenovirus death protein (ADP), encoded within the E3 region, is maintained (i.e., contained) in the adenovirus vector. The ADP
gene, under control of the major late promoter (MLP), appears to code for a protein (ADP) that is important in expediting host cell lysis. Tollefson et al. (1996) J. Virol.
70(4):2296; Tollefson et al. (1992) J. Virol. 66(6):3633. Thus, adenoviral vectors containing the ADP gene may render the adenoviral vector more potent, making possible more effective treatment and/or a lower dosage requirement.
Accordingly, in one embodiment the invention provides adenovirus vectors in which an adenovirus gene is under transcriptional control of a first transactivator regulated transcriptional regulatory element and a polynucleotide sequence encoding an ADP under control of a second transactivator regulated transcriptional regulatory element, and wherein preferably the adenovirus gene is essential for replication. A DNA sequence encoding an ADP and the amino acid sequence of an ADP are depicted in SEQ ID N0:10 and SEQ
ID

N0:11, respectively. Briefly, an ADP coding sequence is obtained preferably from Ad2 (since this is the strain in which ADP has been more fully characterized) using techniques known in the art, such as PCR. Preferably, the Y leader (which is an important sequence for correct expression of late genes) is also obtained and ligated to the ADP
coding sequence. The ADP coding sequence (with or without the Y leader) can then be introduced into the adenoviral genome, for example, in the E3 region (where the ADP
coding sequence will be driven by the MLP). The ADP coding sequence could also be inserted in other locations of the adenovirus genome, such as the E4 region. Alternatively, the ADP coding sequence could be operably linked to a different type of TRE, including, but not limited to, another viral TRE.
It is understood that the present invention does not exclude oncolytic vectors containing additional genes under control of transactivator regulated transcriptional regulatory elements. Accordingly, the invention provides viral vectors comprising a third gene under transcriptional control of a third TRE. The third THE may or may not be substantially identical to the first and/or second TA- TREs, with all three TREs functional in the same cell. Preferably, the third gene is one that contributes to cytotoxicity (whether direct and/or indirect), more preferably one that contributes to and/or enhances cell death.
Delivery ~f ~ncolytic ~/ectors To Cells The oncolytic vectors can be used in a variety of forms, including, but not limited to, naked polynucleotide (usually DNA) constructs; polynucleotide constructs complexed with agents to facilitate entry into cells, such as cationic liposomes or other compounds such as polylysine; packaged into infectious adenovirus particles (which may render the adenoviral vectors) more immunogenic); packaged into other particulate viral forms such as HSV or AAV; complexed with agents to enhance or dampen an immune response; complexed with agents that facilitate in vivo transfection, such as DOTMATM, D~TAPT"', and polyamines.
If an oncolytic vector is packaged into a virus, the virus itself may be selected to further enhance targeting. For example for an adenoviral vector, adenovirus fibers mediate primary contact with cellular receptors) aiding in tropism. See, e.g., Arnberg et al. (1997) Virol. 227:239-244. If a particular subgenus of an adenovirus serotype displayed tropism for a target cell type and/or reduced affinity for non-target cell types, such a subgenus (or subgenera) could be used to further increase cell-specificity of cytotoxicity and/or cytolysis.
Adenovirus fiber, hexon or other surface proteins may be modified to enhance the specificity of uptake by target cells.
The modified oncolytic vectors may be delivered to the target cell in a variety of ways, depending upon whether the cells are in culture, ex vivo or in vivo. In situations where in vivo delivery is desired, delivery can be achieved in a variety of ways, employing liposomes, direct injection, subcutaneous injection, intramuscular injection, catheters, intravenous inhalation, topical applications, intravenous infusion, etc. Due to the high efficiency of transfection of various oncolytic vectors, one can achieve a large number of modified cells. In the case of neoplasia, where toxins are produced, the toxins may be released locally, so as to affect cells that may not have been transfected. In this manner, one can specifically eliminate neoplastic cells, without a significant effect on the normal cells. In addition, expression of viral proteins will serve to activate the immune system against the target cells. Finally, proliferation of the replication competent viral vector in a host cell will lead to cell death. The means of delivery will depend in large part on the particular vector (including its form) as well as the type and location of the target cells (i.e., whether the cells are in vifir~ or in viv~).
In the example of a packaged virus, e.g., an adenovirus, the adenovirus may be administered in an appropriate physiologically acceptable carrier at a dose of about 10~ to 1 O". The multiplicity of infection will generally be in the range of about 0.001 to 100. The virus may be administered one or more times, depending upon the immune response potential of the host. If necessary, the immune response may be diminished by employing a variety of immunosuppressants or treatments to decrease the level of circulating antibody, so as to permit repetitive administration, without a strong immune response.
If administered as a polynucleotide construct (i.e., not packaged as a virus) about 0.01 micrograms to 1000 micrograms of viral vector can be administered. The oncolytic vector may be administered one or more times, or may be administered as multiple simultaneous injections. Dependent upon the type of replication competent virus employed, such as herpes simplex virus (HS!/), reovirus, vesicular stomatitis virus (!!SV), Newcastle Disease virus, vacinia virus, Vilest Nile virus, coxsackie virus, poliovirus and measles virus, the amount of virus to be administered is based on standard knowledge about that particular virus (which is readily obtainable from, for example, published literature) and can be determined empirically.
In some embodiments, a packaged viral vectors) is complexed to a hydrophilic polymer to create a masked virus. The hydrophilic polymer is attached (covalently or non-covalently) to the capsid proteins of the virus, in the case of adenovirus, particularly the hexon and fiber proteins. In preferred embodiments, the viral vectors of the instant invention are complexed with masking agents to create masked viral vectors.
(See, e.g., US Application Serial No. 10/139,089, published as 20030152553. In the adenoviral vector embodiments of the invention, masked viruses are advantageous due to (a) the masking of the adenovirus surface to adenovirus neutralizing antibodies or opsonins which are in circulation and (b) increasing the systemic circulation time of adenovirus particles by reduction of non-specific clearance mechanisms in the body (i.e. macrophages, etc.). In the in vivo context, the systemic delivery of a masked virus results in a longer circulation of virus particles, less immunogenicity, and increased biodistribution with a decrease in clearance by the liver and spleen.
Host Cells and Target Cells The present invention also provides host cells and target cells comprising (i.e., transformed with) the viral vectors described herein. Host cells include both prokaryotic and eukaryotic host cells as long as sequence requisite for maintenance in that host, such as appropriate replication origin(s), are present. For convenience, selectable markers are also provided. Prokaryotic host include bacterial cells, for example, E. coli and mycobacteria.
Among eukaryotic host cells are yeast, insect, avian, amphibian, plant and mammalian host cells. Numerous host cells are known in the art and need not be described in detail herein.
Suitable target cells for the viral vectors of the invention include any eukaryotic cell type that allows function of the TREs and transactivator regulated transcriptional regulatory elements, preferably mammalian, more preferably human, even more preferably neoplastic cells. Suitable target cells also include any cells that produce proteins and other factors necessary for expression of the gene under control of the TREs, such factors necessary for said expression are produced naturally or recombinantly. For example, if the TRE(s) used is prostafie cell-specific, the cells are preferably prostate cells. The prostate cells used may 2.0 or may not be producing an androgen receptor, depending on whether the promoter used is androgen-inducible. If an androgen-inducible promoter is used, non-androgen receptor producing cells, such as HLF, HLE, and 3T3 and the non-AR-producing prostate cancer cells PC3 and ~1J14.5 can be used, provided an androgen receptor-encoding expression vector is introduced into the cells along with the adenovirus. For example, ifi the oncolytic vector comprises a THE derived from the AFP gene, suitable host cells include any cell type that produces AFP, including but not limited to, Hep3B, HepG2, HuH7, HuH1/C12.
Activity of a given THE in a given cell can be assessed by measuring the level of expression of an operably-linked reporter gene using standard assays. The comparison of expression between cells in which the THE is suspected of being functional and the control cell indicates the presence or absence of transcriptional enhancement.
Comparisons between or among various TREs can be assessed by measuring and comparing levels of expression within a single target cell line. It is understood that absolute transcriptional activity of a THE will depend on several factors, such as the nature of the target cell, delivery mode and form of a TRE, and the coding sequence that is to be selectively transcriptionally activated. To compensate for various plasmid sizes used, activities can be expressed as relative activity per mole of transfected plasmid.
Alternatively, the level of transcription (i.e., mRNA) can be measured using standard Northern analysis and hybridization techniques. Levels of transfection (i.e., transfection efficiencies) are measured by co-transfecting a plasmid encoding a different reporter gene under control of a different TRE, such as the CMV immediate early promoter.
This analysis can also indicate negative regulatory regions, i.e., silencers.
Compositions The present invention also includes compositions, including pharmaceutical compositions, containing the viral vectors described herein. Such compositions are useful for administration in vivo, for example, when measuring the degree of transduction and/or effectiveness of cell killing in an individual. Preferably, these compositions further comprise a pharmaceutically acceptable excipient. These compositions, which comprise an effective amount of a viral vector of the invention in a pharmaceutically acceptable excipient, are suitable for systemic administration to individuals in unit dosage forms, sterile parenteral solutions or suspensions, sterile non-parenteral solutions or oral solutions or suspensions, oil in water or water in oil emulsions and the like. Formulations for parenteral and nonparenteral drug delivery are known in the art and are set forth in Remington's Pharmaceutical Sciences, 18t" Edition, Mack Publishing (1990). Compositions also include lyophilized and/or reconstituted forms of the viral vectors (including those packaged as a virus, such as adenovirus) of the invention.
~ther compositions are used, and are useful for, detection methods described herein. For these compositions, the viral vector usually is suspended in an appropriate solvent or solution, such as a bufFer system. Such solvent systems are well known in the art.
Kits The present invention also encompasses kits containing an oncolytic vector of the invention. These kits can be used for diagnostic and/or monitoring purposes, preferably monitoring. Procedures using these kits can be performed by clinical laboratories, experimental laboratories, medical practitioners, or private individuals. Kits embodied by this invention allow one to detect the presence of target cells in a suitable biological sample, such as biopsy specimens.
The kits of the invention comprise an oncolytic vector as described herein in suitable packaging. The kit may optionally provide additional components that are useful in the procedure, including, but not limited to, buffers, developing reagents, labels, reacting surfaces, means for detection, control samples, instructions, and interpretive information.

Preparation Of The Viral Vectors Of The Invention The viral vectors of this invention can be prepared using recombinant techniques that are standard in the art. Generally, TREs are inserted 5' to the adenoviral and transactivator genes of interest, preferably one or more early genes (although late genes) may be used). TREs can be prepared using oligonucleotide synthesis (if the sequence is known) or recombinant methods (such as PCR and/or restriction enzymes).
Convenient restriction sites, either in the natural DNA sequence or introduced by methods such as PCR
or site-directed mutagenesis, provide an insertion site for the TREs.
Accordingly, convenient restriction sites for annealing (i.e., inserting) TREs can be engineered onto the 5' and 3' ends of the THE using standard recombinant methods, such as PCR.
Polynucleotides used for making the oncolytic viral vectors of the invention may be obtained using standard methods in the art such as chemical synthesis recombinant methods andlor obtained from biological sources.
In the case of adenovirus, the vectors are typically prepared by employing two plasmids, one plasmid providing for the left-hand region of adenovirus and the other plasmid providing for the right hand region, where the two plasmids share at least about 500nt of middle region for homologous recombination. In this way, each plasmid, as desired, may be independently manipulated, followed by cotransfection in a competent host, providing complementing genes as appropriate, or the appropriate transcription factors for initiation of transcription from the PSE for propagation of the adenovirus.
For convenience, plasmids are available that provide the necessary portions of the adenovirus. Plasmid pXC.1 (fVlcKinnon (1982) Gene 19:33-4.2) contains the wild-type left-hand end of AdS, pBHG10 provides the right-hand end of AdS, with a deletion in E3. The deletion in E3 provides room in the virus to insert the 2kb minimal PSE
without deleting the wild-type enhancer-promoter. The gene for E3 is located on the opposite strand from E4 (r-strand).
For manipulation of the early genes, the transcription start site of Ad5 E1A
is at nt 560 and the ATG start site of the E1A protein is at nt 610 in the virus genome. This region can be used for insertion of the cell specific element, e.g., PSE.
Conveniently, a restriction site may be introduced by employing the polymerase chain reaction (PCR), where the primer that is employed may be limited to the Ad5 genome, or may involve a portion of the plasmid carrying the Ad5 genomic DNA. For example, where pBR322 is the backbone, the primers may use the EcoRl site in the pBR322 backbone and the Xpal site at nt 1339 of AdS. By carrying out the PCR in two steps, where overlapping primers at the center: of the region introduce a sequence change resulting in a unique restriction site, one can provide for insertion of the cell specific response element at that site.

A similar strategy may also be used for insertion of the cell specific response element to regulate E1 B. The E1 B promoter of Ad5 consists of a single high-affinity recognition site for Spl and a TATA box. This region extends from 1636 to 1701 nt. By insertion of the cell specific response element in this region, one can provide for cell specific transcription of the E1 B gene. By employing the left-hand region modified with the cell specific response element regulating E1A, as the template for introducing the cell specific response element to regulate E1 B, the resulting adenovirus will be dependent upon the cell specific transcription factors for expression of both E1A and E1 B.
For example, we have introduced an Agel site 12 by 5' to the E1A initiation colon (Ad5 nucleotide 547) by oligo-directed mutagenesis and linked PCR. In addition, an Eagl site was created upstream of the E1 B start site by inserting a G residue at Ad5 nt 1682 by oligonucleotide directed mutagenesis. To simplify insertion of a THE in the Eagl site, the endogenous Eagl site in CN95 was removed by digestion with Eagl, treatment with mung bean nuclease, and religation to construct CN114. In this way, we generated an adenovirus vector containing unique Agel and Eagl sites in the proximal upstream region to E1A and E1 B, respectively. Using these unique sites, one can insert a THE which has engineered Agel or Eagl sites, thus simplifying construction of recombinant adenovirus vectors.
Accordingly, the invention includes an adenoviral vector comprising a unique Agel site 5' of the E1A initiation colon and a unique Eagl site 5' of E1B.
Similarly, a THE may be inserted upsfiream of the E2 gene. The E2 early promoter, mapping in Ad5 from 27050-27150, consists of a major and a minor transcription initiation site, the latter accounting for about 5°/~ of the E2 transcripts, two non-canonical TATA
boxes, two E2F transcription factor binding sites and an ATF transcription factor binding site (for a detailed review of the E2 promoter architecture see Swaminathan et al., Curr. Topics in ntlicrobiol. and Immunol. (1995) 199 part 3:177-194).
The E2 late promoter overlaps with the coding sequences of a gene encoded by the counterstrand and is therefore not amenable to genetic manipulation. However, the E2 early promoter overlaps only for a few base pairs with sequences coding for a 33-kD protein on the counterstrand. Notably, the Spel restriction site (Ad5 position 27082) is part of the stop colon for the above mentioned 33 kD protein and conveniently separates the major E2 early transcription initiation site and TATA-binding protein site from the upstream transcription factor biding sites E2F and ATF. Therefore, insertion of a THE
having Spel ends into the Spel site in the plus-strand would disrupt the endogenous E2 early promoter of Ad5 and should allow THE regulated expression of E2 transcripts.
For E4, one must use the right hand portion of the adenovirus genome. The E4 transcription start site is predominantly at nt 35609, the TATA box at nt 35638 and the first AUG/GUG of ORF1 is at nt 35532. Virtanen et al. (1984) J. Virol. 51: 822-831.
Using any of the above strategies for the other genes, a heterologous THE may be introduced upstream from the transcription start site. For the construction of mutants in the E4 region, the co-transfection and homologous recombination are performed in W162 cells (Vlleinberg et al.
(1983) Proc. Natl. Acad. Sci. USA 80:5383-5386) which provide E4 proteins in trans to complement defects in synthesis of these proteins.
Methods of packaging adenovirus polynucleotides into adenovirus particles are known in the art and are described in the Examples.
Methods Using The Oncolytic Vectors Of The Invention The subject oncolytic vectors can be used for a wide variety of purposes, which will vary with the desired or intended result. Accordingly, the present invention includes methods using the oncolytic viral vectors described above. In one embodiment, methods for using oncolytic vectors comprise introducing the vector into a cell, preferably a eukaryotic cell, more preferably a mammalian cell, in vitro or in vivo. In one preferred embodiment, an oncolytic vector of the invention is administered in vivo for treatment of cancer.
Purposes for introducing transient expression include indications that may be treated involving undesired proliferation other than tumors, such as psoriatic lesions, restenosis, wound healing, tissue repair, enhanced immune response, resistance to infection, production of factors, enhanced proliferation, investigation of metabolic or other physiological pathways, comparison of activity of cells in fihe presence and absence of the virus introduced transgene, by comparing the activity of the cell before, during and after the modification with the virus, etc. The subject vectors can be used to free a mixture of cells of a particular group of cells, where the group of cells is the target cells. By having the oncolytic virus be selectively competent for propagation in the target cells, only those cells will be killed on proliferation of the oncolytic virus. By combining the virus with the mixture of cells, for example, in culture or in vivo, the oncolytic virus will only be capable of proliferation in the target cells, and will be regulated by the presence of the inducing agent.
In this way cells other than the target cells will not be affected by the oncolytic virus, while the target cells will be killed. The expansion of the oncolytic virus due to propagation in the target cells will ensure that the mixture is substantially freed of the target cells. Once the target cells are destroyed, the oncolytic virus will no longer be capable of propagation, but in culture may be retained so as to continually monitor the mixture for recurrence of the target cell, e.g., a mutated cell or neoplastic cell. The presence or concentration of an inducing agent and/or condition provides further control for viral replication.
By identifying genes that are expressed specifically by the target host cells, based on the nature of the cells, their level of maturity or their condition, the target cell specific response element can be used to provide genetic capability to such cells, where the genetic capability will be absent in other cells, even when transfected with the oncolytic virus.
In one embodiment, methods for using oncolytic virus vectors comprise introducing an oncolytic virus vector into a target cell, preferably a neoplastic cell. In another embodiment, methods for using oncolytic virus vectors comprise introducing an oncolytic virus vector into a prostate cell. In another embodiment, methods for using oncolytic virus vectors comprise introducing an oncolytic virus vector into a liver cell. In another embodiment, methods for using oncolytic virus vectors comprise introducing an oncolytic virus vector into a breast cancer cell. In another embodiment, methods for using oncolytic virus vectors comprise introducing an oncolytic virus vector into a colon cancer cell.
In one embodiment, methods are provided for conferring selective cytotoxicity in cells which allow function of the TRE, comprising contacting cells with an oncolytic virus vector described herein, such that the oncolytic virus vectors) enters, i.e., transduces the cell(s). Cytotoxicity can be measured using standard assays in the art, such as dye exclusion, 3H-thymidine incorporation, and/or lysis.
In another embodiment, methods are provided for propagating an oncolytic virus specific for cells which allow function of the cell type-specific and transactivator regulated transcriptional regulatory element(s), preferably eukaryotic cells, more preferably mammalian cells. These methods entail combining an oncolytic virus vector with mammalian cells that allow function of the TREs, whereby said oncolytic virus is propagated.
Another embodiment provides methods of killing cells that allow a THE to function (i.e., target cells) comprising combining the mixture of cells with an oncolytic virus vector of the present invention. The mixture of cells is generally a mixture of cancerous cells in which the TREs are functional and normal cells, and can be an in vivo mixture or in vitro mixture.
The invention also includes methods for detecting cells in which a CT-THE
andlor transactivator regulated transcriptional regulatory element is functional in a biological sample. These methods are particularly useful for monitoring the clinical and/or physiological condition of an individual (i.e., mammal), whether in an experimental or clinical setting. For these methods, cells of a biological sample are contacted with an oncolytic virus vector, and replication of the oncolytic viral vector is detected. A
suitable biological sample is one in which target cells may be or are suspected to be present.
Generally, in mammals, a suitable clinical sample is one in which target cancerous cells are suspected to be present. Such cells can be obtained, for example, by needle biopsy or other surgical procedure. Cells to be contacted may be treated to promote assay conditions such as selective enrichment and/or solubilization. In these methods, target cells can be detected using in vitro assays that detect proliferation, which are standard in the art. Examples of such standard assays include, but are not limited to, burst assays (which measure virus yields) and plaque assays (which measure infectious particles per cell). Also, propagation can be detected by measuring specific oncolytic viral DNA replication, which are also standard assays.
The invention also provides methods of modifying the genotype of a target cell, comprising contacting the target cell with an oncolytic virus vector described herein, wherein the oncolytic viral vector enters the cell. , The invention further provides methods of suppressing tumor cell growth, comprising contacting a tumor cell with an oncolytic viral vector of the invention such that the oncolytic viral vector enters the tumor cell and exhibits selective cytotoxicity for the tumor cell. Tumor cell growth can be assessed by any means known in the art, including, but not limited to, measuring tumor size, determining whether tumor cells are proliferating using a 3H-thymidine incorporation assay, or counting tumor cells. "Suppressing" tumor cell growth means any or all of the following states: slowing, delaying, and stopping tumor growth, as well as tumor shrinkage. "Suppressing" tumor growth indicates a growth state that is curtailed when compared to growth without contact with, i.e., transfection by, an oncolytic viral vector described herein.
The invention also provides methods of lowering the levels of a tumor cell marker in an individual, comprising administering to the individual an oncolytic viral vector of the present invention, wherein the oncolytic viral vector is selectively cytotoxic in cells producing the tumor cell marker. Tumor cell markers include, but are not limited to, PSA, GEA and hl<2. Methods of measuring the levels of a tumor cell marker are known to those of ~rdinary skill in the art and include, but are not limited to, immunological assays, such as enzyme-linked immunosorbent assay (ELISA), using antibodies specific for the tumor cell marleer. In general, a biological sample is obtained from the individual to be tested, and a suitable assay, such as an ELISA, is performed on the biological sample.
The invention also provides methods of treatment, in which an effective amount of an oncolytic viral vector described herein is administered to an individual.
For example, treatment using an oncolytic viral vector in which at least one cell type-specific THE is specific for prostate cells (e.g., PSE TRE, PB-TRE, and/or hKLK2-TRE) is indicated in individuals with prostate-associated diseases as described above, such as hyperplasia and cancer. In this example, also indicated are individuals who are considered to be at risk for developing prostate-associated diseases, such as those who have had disease which has been resected and those who have had a family history of prostate-associated diseases.
Determination of suitability of administering oncolytic viral vectors) of the invention will depend, inter alia, on assessable clinical parameters such as serological indications and histological examination of tissue biopsies. Generally, a pharmaceutical composition comprising an oncolytic viral vector is administered. Pharmaceutical compositions are described above.
The following examples are offered by way of illustration and not by way of limitation.

T_ etracycline Regulated Oncolytic Virus Replication Specific for Prostate Tissue A prostate-specific oncolytic adenovirus is provided in which replication is regulated using the Tet-On system to control expression of at least one adenoviral gene.
In the case of the Tet-On regulated gene expression system to control oncolytic viral replication, the expression of the immediate early adenoviral E1A gene region is placed under the control of the chimeric promoter element - the tetracycline responsive element (Fig. 1 ) by removal of the endogenous adenoviral promoter elements and insertion of the tetracycline responsive element. An expression cassette for production of the modified reverse tetracycline transactivator (rtTA(2)s-M2), which binds to the tetracycline responsive element to induce expression in the presence of tetracycline and its derivatives (such as Doxycycline), is placed in the E3 region of the virus under the control of a tissue/tumor specific promoter (a CT-TRE), exemplified herein by the human prostate specific antigen (PSA) promoter. In this manner, expression of the rtTA transactivator (a TA-TRE) is limited to the tissue/or tumor in which the PSA promoter is active e.g. prostate-derived tissue. lJpon the addition of tetracycline (or a derivative thereof) the rtTA transactivator binds to the tetracycline responsive element and switches on E1A transcription and thus expression. From expression of the E1A profiein, the adenovirus replication process continues, resulting in the eventual death of the host cell and release of further adenoviral progeny. In comparison, in the absence of the inducer or the virus entering a non-target cell, rtTA
cannot bind to the tetracycline responsive element, E1A proteins are not expressed and viral replication does not continue. The E1A gene region is described in this example, however, any essential ORF of adenovirus can be regulated in this manner, i.e. the tetracycline responsive element can control the E1 a, E1 b, E2 or E4 region. Furthermore multiple ORFs may be placed under tetracycline regulated control i.e. E1 and E4, E1 and E2, to increase the control of the system.
_1A Ad5 with PSE Driving Expression of E1A
The cloning and characterization of a minimal prostate-specific enhancer (PSE) is described in Schuur et al., 1996. Plasmid CN71 contains a minimal PSE (from-5322 by to -3875bp relative to the transcription start site of the PSA gene) and -532 to +11 of the PSA
promoter. CN71 was cut with Xhol/Hindlll which removes the PSA promoter. A
shorter promoter, from -230 to +7 was amplified by PCR. The PCR product was cut with Xhol/Hindlll and ligated back into Xhol/Hindlll cut CN71 creating CN105. The plasmid, pUHrt62-1 (2) (a generous gift from W. Hillen) was then cut with EcoRl, the site blunted by klenow fragment, and then cut with Xho I to remove the CMV promoter from the construct.
The PSA promoter fragment from above was then placed 5' of the rtTA2(s)-M2 transgene (in pUHrt62-1 (2)) in place of CMV to create pUH-PSA-rt62-1 (2). The PSA-rtTA2(s)-M2 fragment (incorporating the SV40 Late polyA) was then liberated from pUH-PSA-rt62-1 (2) using Xho I / Hind III digestion and then ligated /inserted via Xba I
restriction sites into of pABS4 (Microbix, Toronto), a shuttle plasmid containing the kanamycin-resistance, gene to create pABS4-PSA-rtTA. By digesting pABS4-PSA-rtTA with Pacl, a fragment containing the FCan~ gene and the PSA-rtTA2(s)-M2 expression cassette was isolated and ligated into similarly cut BHG11 (Microbix), which contains a unique Pacl site engineered in the E3 region of Ad5 to create BHG11-PSA-rtTA-kan~. The kanR gene was removed by digesting BHG11-PSA-rtTA-kanR with Swal and religating the vector (BHG11-PSA-rfiTA).
1 B Attenuated Ad5 with Tetracycline responsive promoter Driving E1A and Retaining the Endoctenous Ad5 E1A Promoter and Enhance In the absence of a functional E1A gene, viral infection does not proceed for the gene products necessary for viral DNA replication are not produced (Nevins (1939) Adv.
Virus Res. 31:35-31). The transcription start site of Ad5 E1A is at nt 560 and the ATG start site of the E1A protein is at nt 610 in the virus genome.
pXC.1 was purchased from Microbix Biosystems Inc. (Toronto). pXC.1 contains Adenovirus 5 sequences from bp22 to 5790. An Agel site was introduced 12 by 5' to the E1A initiation codon (Ad5 nucleotide 547) by oligo-directed mutagenesis and linked PCR.
The plasmid pXC.1 was PCR amplified using primers confiaining an extra A to introduce an Agel site. This created a segment from the EcoRl site in the pBR322 backbone to Ad5 nt 560. A second segment of pXC.1 from Ad nucleotide 541 to the Xbal site at Ad nucleotide 1339 was amplified using primers containing an extra T to introduce an Agel site. A mixture of these two PCR amplified DNA segments was mixed and amplified with primers 3 and 4 to create a DNA segment from the EcoRl site to the Xbal site of pXC.1. This DNA
segment encompasses the leftmost 1317 bases of Adenovirus sequence and contained an Agel site at Ad nucleotide 547. This DNA segment was used to replace the corresponding segment of pXC.1 to create CN95. Similarly, a Tetracycline responsive element with Agel ends was PCR amplified from the plasmid pTRE2 (purchased from BD Biosciences Clontech) to create pXC-TRE-E1 a .

1 C. The virus created by homologous recombination - CG1974 An Ad5 recombinant virus containing the PSA promoter driving rtTA(2)s-M2 gene in the E3 region and the tetracycline responsive element driving E1a in the E1 region was thus constructed. Virus was generated by homologous recombination in low passage 293 cells, a human kidney cell line that expresses Ad E1A and E1B proteins. This was accomplished by co-transfection of pXC-TRE-E1a and BHG11-PSA-rtTA. Genomic integrity of the resulting recombinant virus construct was verified using Hind III digestion and was designated CG1974 (Figure 1).

In an further example, an oncolytic vector is "armed" with a therapeutic transgene to increase efficacy, e.g., granulocyte macrophage colony stimulating factor (GMCSF) or thymidine kinase (TK). The expression of these therapeutic transgenes may be placed under the control of the regulated gene expression system. In this manner, the inducer used within the regulated gene expression system switches on both virus replication and therapeutic gene expression at the same time. An example of such a vector is given in Figure 2 which illustrates a prostate specific oncolytic viral vector armed with GMCSF with tetracycline regulated replication and expression control. In this example, a tetracycline responsive element (TRE) drives E1 and GMGSF expression. An IRES allows 2 coding sequences to be expressed from a single promoter and a prostate specific antigen (PSA) promoter drives expression of the reverse tet-responsive transactivator (rtTA). Here, the vector functions just as set forth above, with both E1 and GMCSF expression switched on by the addition of Tet to the system. In this example, an internal ribosome entry site (IRES) or 2A sequence allows 2 coding regions to be expression using a single promoter.

A number of variant tetracycline regulated gene control systems for use in the manner described above. For example, the tetracycline controlled transcriptional silencer (tTS) system (Freundlieb et al 1999) in conjunction with the Tet-On system described above may be used to more tightly control gene expression (see Figure 3 which illustrates an oncolytic viral vector with dual tetracycline regulated replication control for use in bladder cancer). In this example a tetracycline responsive element (TRE) drives E1 gene expression and a human uroplakin II (hUPll) promoter drives expression of the reverse tet-responsive transactivator (rtTA) and tet-controlled transcriptional silencer (tTS). The use of the tTS repressor molecule, in addition to the rtTA transactivator, decreases basal or "leaky"
gene expression in the "ofP' state to increase the degree of gene regulation.
For example the system in Figure 3 expresses both the Tet-on transactivator and tTs repressor from the human uroplakin II promoter using an internal ribosome entry site (IRES). In this manner, expression of the rtTA and tTS are specific to the bladder tissue cell type.
The expression of the essential E1 gene is again under the control of the TRE. In the absence of Tet the tTS
molecule binds to the THE element and actively represses E1 gene expression.
This active repression by the tTS molecule may be required since adenovirus has a number of transcription enhancer elements present within its genome which could cause "leaky"
expression of the E1 promoter, even in the absence of its endogenous promoter, leading to a limited amount of virus formation in the absence of the inducer. Upon the addition of tetracycline, a conformational change in the tTS molecule will prevent binding to and active repression of expression from the TRE. The rtTA, Tet-~n transactivator, would then be able to induce expression of E1 by the resulting ability to bind to the TRE, such that replication proceeds.
An alternative system is presented in Figure 4 which represents an example of an oncolytic viral vector with rapamycin (ARIAD system) regulated replication control for use in pan-cancer applications. This system relies on a chimeric promoter encompassing 8 copies of the recognition site for ZFHD1 upstream of a minimal IL-2 promoter driving E1 gene expression and a human E2F promoter (E2F) driving expression of the "activation domain"
(the rapamycin binding domain of FRAP (FRB) fused to the activation domain of p65 sub-unit of NF-I<B) and "DNA binding domain" (which encodes a chimeric DNA-binding molecule with ZFHD1 fused to 3 copies of FhCBP) It is evident from the above description that regulatable replication-competent viruses can be provided as vehicles specific for parfiicular host cells, where the viruses selectively replicate in particular target cells and viral replication may be regulated.
All publications and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.
The invention now being fully described, it will be apparent to one of ordinary skill in the art that many changes and modifications can be made thereto without departing from the spirit or scope of the appended claims.

SEQUENCE LISTING
<110> Harding, Thomas Yu, De Chao <120> SYSTEM FOR EXTERNAL CONTROL OF ONCOLYTIC VIRUS REPLICATION
<210> 1 <211> 5836 <212> DNA
<213> Homo Sapiens <400> 1 aagcttctag ttttcttttc ccggtgacat cgtggaaagc actagcatct ctaagcaatg 60 atctgtgaca atattcacag tgtaatgcca tccagggaac tcaactgagc cttgatgtcc 120 agagattttt gtgttttttt ctgagactga gtctcgctct gtgccaggct ggagtgcagt 180 ggtgcaacct tggctcactg caagctccgc ctcctgggtt cacgccattc tcctgcctca 240 gcctcctgag tagctgggac tacaggcacc cgccaccacg cctggctaat ttttttgtat 300 ttttagtaga gatggggttt cactgtgtta gccaggatgg tctcagtctc ctgacctcgt 360 gatctgccca ccttggcctc ccaaagtgct gggatgacag gcgtgagcca ccgcgcctgg 420 ccgatatcca gagatttttt ggggggctcc atcacacaga catgttgact gtcttcatgg 480 ttgactttta gtatccagcc cctctagaaa tctagctgat atagtgtggc tcaaaacctt 540 cagcacaaat cacaccgtta gactatctgg tgtggcccaa accttcaggt gaacaaaggg 600 actctaatct ggcaggatat tccaaagcat tagagatgac ctcttgcaaa gaaaaagaaa 660 tggaaaagaa aaagaaagaa aggaaaaaaa aaaaaaaaaa gagatgacct ctcaggctct 720 gaggggaaac gcctgaggtc tttgagcaag gtcagtcctc tgttgcacag tctccctcac 780 agggtcattg tgacgatcaa atgtggtcac gtgtatgagg caccagcaca tgcctggctc 840 tggggagtgc cgtgtaagtg tatgcttgca ctgctgaatg cttgggatgt gtcagggatt 900 atcttcagca cttacagatg ctcatctcat cctcacagca tcactatggg atgggtatta 960 ctggcctcat ttgatggaga aagtggctgt ggctcagaaa ggggggacca ctagaccagg 1020 gacactctgg atgctgggga ctccagagac catgaccact caccaactgc agagaaatta 1080 attgtggcct gatgtccctg tcctggagag ggtggaggtg gaccttcact aacctcctac 1140 cttgaccctc tcttttaggg ctctttctga cctccaccat ggtactagga ccccattgta 1200 ttctgtaccc tcttgactct atgaccccca ctgcccactg catccagctg ggtcccctcc 1260 tatctctatt cccagctggc cagtgcagtc tcagtgccca cctgtttgtc agtaactctg 1320 aaggggctga cattttactg acttgcaaac aaataagcta actttccaga gttttgtgaa 1380 tgctggcaga gtccatgaga ctcctgagtc agaggcaaag gcttttactg ctcacagctt 1440 agcagacagc atgaggttca tgttcacatt agtacacctt gcccccccca aatcttgtag 1500 ggtgaccaga gcagtctagg tggatgctgt gcagaagggg tttgtgccac tggtgagaaa 1560 cctgagatta ggaatcctca atcttatact gggacaactt gcaaacctgc tcagcctttg 1620 tctctgatga agatattatc ttcatgatct tggattgaaa acagacctac tctggaggaa 1680 catattgtat cgattgtcct tgacagtaaa caaatctgtt gtaagagaca ttatctttat 1740 tatctaggac agtaagcaag cctggatctg agagagatat catcttgcaa ggatgcctgc 1800 tttacaaaca tccttgaaac aacaatccag aaaaaaaaag gtgttgctgt ctttgctcag 1860 aagacacaca gatacgtgac agaaccatgg agaattgcct cccaacgctg ttcagccaga 1920 gccttccacc cttgtctgca ggacagtctc aacgttccac cattaaatac ttcttctatc 1980 acatcctgct tctttatgcc taaccaaggt tctaggtccc gatcgactgt gtctggcagc 2040 actccactgc caaacccaga ataaggcagc gctcaggatc ccgaaggggc atggctgggg 2100 atcagaactt ctgggtttga gtgaggagtg ggtccaccct cttgaatttc aaaggaggaa 2160 gaggctggat gtgaaggtac tgggggaggg aaagtgtcag ttccgaactc ttaggtcaat 2220 gagggaggag actggtaagg tcccagctcc cgaggtactg atgtgggaat ggcctaagaa 2280 tctcatatcc tcaggaagaa ggtgctggaa tcctgagggg tagagttctg ggtatatttg 2340 tggcttaagg ctctttggcc cctgaaggca gaggctggaa ccattaggtc cagggtttgg 2400 ggtgatagta atgggatctc ttgattcctc aagagtctga ggatcgaggg ttgcccattc 2460 ttccatcttg ccacctaatc cttactccac ttgagggtat caccagccct tctagctcca 2520 tgaaggtccc ctgggcaagc acaatctgag catgaaagat gccccagagg ccttgggtgt 2580 catccactca tcatccagca tcacactctg agggtgtggc cagcaccatg acgtcatgtt 2640 gctgtgacta tccctgcagc gtgcctctcc agccacctgc caaccgtaga gctgcccatc 2700 ctcctctggt gggagtggcc tgcatggtgc caggctgagg cctagtgtca gacagggagc 2760 ctggaatcat agggatccag gactcaaaag tgctagagaa tggccatatg tcaccatcca 2820 tgaaatctca agggcttctg ggtggagggc acagggacct gaacttatgg tttcccaagt 2880 ctattgctct cccaagtgag tctcccagat acgaggcact gtgccagcat cagccttatc 2940 tccaccacat cttgtaaaag gactacccag ggccctgatg aacaccatgg tgtgtacagg 3000 agtagggggt ggaggcacgg actcctgtga ggtcacagcc aagggagcat catcatgggt 3060 ggggaggagg caatggacag gcttgagaac ggggatgtgg ttgtatttgg ttttctttgg 3120 ttagataaag tgctgggtat aggattgaga gtggagtatg aagaccagtt aggatggagg 3180 atcagattgg agttgggtta gataaagtgc tgggtatagg attgagagtg gagtatgaag 3240 accagttagg atggaggatc agattggagt tgggttagag atggggtaaa attgtgctcc 3300 ggatgagttt gggattgaca ctgtggaggt ggtttgggat ggcatggctt tgggatggaa 3360 atagatttgt tttgatgttg gctcagacat ccttggggat tgaactgggg atgaagctgg 3420 gtttgatttt ggaggtagaa gacgtggaag tagctgtcag atttgacagt ggccatgagt 3480 tttgtttgat ggggaatcaa acaatggggg aagacataag ggttggcttg ttaggttaag 3540 ttgcgttggg ttgatggggt cggggctgtg tataatgcag ttggattggt ttgtattaaa 3600 ttgggttggg tcaggttttg gttgaggatg agttgaggat atgcttgggg acaccggatc 3660 catgaggttc tcactggagt ggagacaaac ttcctttcca ggatgaatcc agggaagcct 3720 taattcacgt gtaggggagg tcaggccact ggctaagtat atccttccac tccagctcta 3780 agatggtctt aaattgtgat tatctatatc cacttctgtc tccctcactg tgcttggagt 3840 ttacctgatc actcaactag aaacagggga agattttatc aaattctttt tttttttttt 3900 ttttttttga gacagagtct cactctgttg cccaggctgg agtgcagtgg cgcagtctcg 3960 gctcactgca acctctgcct cccaggttca agtgattctc ctgcctcagc ctcctgagtt 4020 gctgggatta caggcatgca gcaccatgcc cagctaattt ttgtattttt agtagagatg 4080 gggtttcacc aatgtttgcc aggctggcct cgaactcctg acctggtgat ccacctgcct 4140 cagcctccca aagtgctggg attacaggcg tcagccaccg cgcccagcca cttttgtcaa 4200 attcttgaga cacagctcgg gctggatcaa gtgagctact ctggttttat tgaacagctg 4260 aaataaccaa ctttttggaa attgatgaaa tcttacggag ttaacagtgg aggtaccagg 4320 gctcttaaga gttcccgatt ctcttctgag actacaaatt gtgattttgc atgccacctt 4380 aatctttttt tttttttttt taaatcgagg tttcagtctc attctatttc ccaggctgga 4440 gttcaatagc gtgatcacag ctcactgtag ccttgaactc ctggccttaa gagattctcc 4500 tgcttcggtc tcccaatagc taagactaca gtagtccacc accatatcca gataattttt 4560 aaattttttg gggggccggg cacagtggct cacgcctgta atcccaacac catgggaggc 4620 tgagatgggt ggatcacgag gtcaggagtt tgagaccagc ctgaccaaca tggtgaaact 4680 ctgtctctac taaaaaaaaa aaaaatagaa aaattagccg ggcgtggtgg cacacggcac 4740 ctgtaatccc agctactgag gaggctgagg caggagaatc acttgaaccc agaaggcaga 4800 ggttgcaatg agccgagatt gcgccactgc actccagcct gggtgacaga gtgagactct 4860 gtctcaaaaa aaaaaaattt tttttttttt tttgtagaga tggatcttgc tttgtttctc 4920 tggttggcct tgaactcctg gcttcaagtg atcctcctac cttggcctcg gaaagtgttg 4980 ggattacagg cgtgagccac catgactgac ctgtcgttaa tcttgaggta cataaacctg 5040 gctcctaaag gctaaaggct aaatatttgt tggagaaggg gcattggatt ttgcatgagg 5100 atgattctga cctgggaggg caggtcagca ggcatctctg ttgcacagat agagtgtaca 5160 ggtctggaga acaaggagtg gggggttatt ggaattccac attgtttgct gcacgttgga 5220 ttttgaaatg ctagggaact ttgggagact catatttctg ggctagagga tctgtggacc 5280 acaagatctt tttatgatga cagtagcaat gtatctgtgg agctggattc tgggttggga 5340 gtgcaaggaa aagaatgtac taaatgccaa gacatctatt tcaggagcat gaggaataaa 5400 agttctagtt tctggtctca gagtggtgca gggatcaggg agtctcacaa tctcctgagt 5460 gctggtgtct tagggcacac tgggtcttgg agtgcaaagg atctaggcac gtgaggcttt 5520 gtatgaagaa tcggggatcg tacccacccc ctgtttctgt ttcatcctgg gcatgtctcc 5580 tctgcctttg tcccctagat gaagtctcca tgagctacaa gggcctggtg catccagggt 5640 gatctagtaa ttgcagaaca gcaagtgcta gctctccctc cccttccaca gctctgggtg 5700 tgggaggggg ttgtccagcc tccagcagca tggggagggc cttggtcagc ctctgggtgc 5760 cagcagggca ggggcggagt cctggggaat gaaggtttta tagggctcct gggggaggct 5820 ccccagcccc aagctt 5836 <210> 2 <211> 5835 <212> DNA
<213> Homo Sapiens <400> 2 aagcttctag ttttcttttc ccggtgacat cgtggaaagc actagcatct ctaagcaatg 60 atctgtgaca atattcacag tgtaatgcca tccagggaac tcaactgagc cttgatgtcc 120 agagattttt gtgttttttt ctgagactga gtctcgctct gtgccaggct ggagtgcagt 180 ggtgcaacct tggctcactg caagctccgc ctcctgggtt cacgccattc tcctgcctca 240 gcctcctgag tagctgggac tacaggcacc cgccaccacg cctggctaat ttttttgtat 300 ttttagtaga gatggggttt cactgtgtta gccaggatgg tctcagtctc ctgacctcgt 360 gatctgccca ccttggcctc ccaaagtgct gggatgacag gcgtgagcca ccgcgcctgg 420 ccgatatcca gagatttttt ggggggctcc atcacacaga catgttgact gtcttcatgg 480 ttgactttta gtatccagcc cctctagaaa tctagctgat atagtgtggc tcaaaacctt 540 cagcacaaat cacaccgtta gactatctgg tgtggcccaa accttcaggt gaacaaaggg 600 actctaatct ggcaggatac tccaaagcat tagagatgac ctcttgcaaa gaaaaagaaa 660 tggaaaagaa aaagaaagaa aggaaaaaaa aaaaaaaaaa gagatgacct ctcaggctct 720 gaggggaaac gcctgaggtc tttgagcaag gtcagtcctc tgttgcacag tctccctcac 780 agggtcattg tgacgatcaa atgtggtcac gtgtatgagg caccagcaca tgcctggctc 840 tggggagtgc cgtgtaagtg tatgcttgca ctgctgaatg gctgggatgt gtcagggatt 900 atcttcagca cttacagatg ctcatctcat cctcacagca tcactatggg atgggtatta 960 ctggcctcat ttgatggaga aagtggctgt ggctcagaaa ggggggacca ctagaccagg 1020 gacactctgg atgctgggga ctccagagac catgaccact caccaactgc agagaaatta 1080 attgtggcct gatgtccctg tcctggagag ggtggaggtg gaccttcact aacctcctac 1140 cttgaccctc tcttttaggg ctctttctga cctccaccat ggtactagga ccccattgta 1200 ttctgtaccc tcttgactct atgaccccca ccgcccactg catccagctg ggtcccctcc 1260 tatctctatt cccagctggc cagtgcagtc tcagtgccca cctgtttgtc agtaactctg 1320 aaggggctga cattttactg acttgcaaac aaataagcta actttccaga gttttgtgaa 1380 tgctggcaga gtccatgaga ctcctgagtc agaggcaaag gcttttactg ctcacagctt 1440 agcagacagc atgaggttca tgttcacatt agtacacctt gcccccccca aatcttgtag 1500 ggtgaccaga gcagtctagg tggatgctgt gcagaagggg tttgtgccac tggtgagaaa 1560 cctgagatta ggaatcctca atcttatact gggacaactt gcaaacctgc tcagcctttg 1620 tctctgatga agatattatc ttcatgatct tggattgaaa acagacctac tctggaggaa 1680 catattgtat cgattgtcct tgacagtaaa caaatctgtt gtaagagaca ttatctttat 1740 tatctaggac agtaagcaag cctggatctg agagagatat catcttgcaa ggatgcctgc 1800 tttacaaaca tccttgaaac aacaatccag aaaaaaaaag gtgttactgt ctttgctcag 1860 aagacacaca gatacgtgac agaaccatgg agaattgcct cccaacgctg ttcagccaga 1920 gccttccacc ctttctgcag gacagtctca acgttccacc attaaatact tcttctatca 1980 catcccgctt ctttatgcct aaccaaggtt ctaggtcccg atcgactgtg tctggcagca 2040 ctccactgcc aaacccagaa taaggcagcg ctcaggatcc cgaaggggca tggctgggga 2100 tcagaacttc tgggtttgag tgaggagtgg gtccaccctc ttgaatttca aaggaggaag 2160 aggctggatg tgaaggtact gggggaggga aagtgtcagt tccgaactct taggtcaatg 2220 agggaggaga ctggtaaggt cccagctccc gaggtactga tgtgggaatg gcctaagaat 2280 ctcatatcct caggaagaag gtgctggaat cctgaggggt agagttctgg gtatatttgt 2340 ggcttaaggc tctttggccc ctgaaggcag aggctggaac cattaggtcc agggtttggg 2400 gtgatagtaa tgggatctct tgattcctca agagtctgag gatcgagggt tgcccattct 2460 tccatcttgc cacctaatcc ttactccact tgagggtatc accagccctt ctagctccat 2520 gaaggtcccc tgggcaagca caatctgagc atgaaagatg ccccagaggc cttgggtgtc 2580 atccactcat catccagcat cacactctga gggtgtggcc agcaccatga cgtcatgttg 2640 ctgtgactat ccctgcagcg tgcctctcca gccacctgcc aaccgtagag ctgcccatcc 2700 tcctctggtg ggagtggcct gcatggtgcc aggctgaggc ctagtgtcag acagggagcc 2760 tggaatcata gggatccagg actcaaaagt gctagagaat ggccatatgt caccatccat 2820 gaaatctcaa gggcttctgg gtggagggca cagggacctg aacttatggt ttcccaagtc 2880 tattgctctc ccaagtgagt ctcccagata cgaggcactg tgccagcatc agccttatct 2940 ccaccacatc ttgtaaaagg actacccagg gccctgatga acaccatggt gtgtacagga 3000 gtagggggtg gaggcacgga ctcctgtgag gtcacagcca agggagcatc atcatgggtg 3060 gggaggaggc aatggacagg cttgagaacg gggatgtggt tgtatttggt tttctttggt 3120 tagataaagt gctgggtata ggattgagag tggagtatga agaccagtta ggatggagga 3180 tcagattgga gttgggttag ataaagtgct gggtatagga ttgagagtgg agtatgaaga 3240 ccagttagga tggaggatca gattggagtt gggttagaga tggggtaaaa ttgtgctccg 3300 gatgagtttg ggattgacac tgtggaggtg gtttgggatg gcatggcttt gggatggaaa 3360 tagatttgtt ttgatgttgg ctcagacatc cttggggatt gaactgggga tgaagctggg 3420 tttgattttg gaggtagaag acgtggaagt agctgtcaga tttgacagtg gccatgagtt 3480 ttgtttgatg gggaatcaaa caatggggga agacataagg gttggcttgt taggttaagt 3540 tgcgttgggt tgatggggtc ggggctgtgt ataatgcagt tggattggtt tgtattaaat 3600 tgggttgggt caggttttgg ttgaggatga gttgaggata tgcttgggga caccggatcc 3660 atgaggttct cactggagtg gagacaaact tcctttccag gatgaatcca gggaagcctt 3720 aattcacgtg taggggaggt caggccactg gctaagtata tccttccact ccagctctaa 3780 gatggtctta aattgtgatt atctatatcc acttctgtct ccctcactgt gcttggagtt 3840 tacctgatca ctcaactaga aacaggggaa gattttatca aattcttttt tttttttttt 3900 tttttttgag acagagtctc actctgttgc ccaggctgga gtgcagtggc gcagtctcgg 3960 ctcactgcaa cctctgcctc ccaggttcaa gtgattctcc tgcctcagcc tcctgagttg 4020 ctgggattac aggcatgcag caccatgccc agctaatttt tgtattttta gtagagatgg 4080 ggtttcacca atgtttgcca ggctggcctc gaactcctga cctggtgatc cacctgcctc 4140 agcctcccaa agtgctggga ttacaggcgt cagccaccgc gcccagccac ttttgtcaaa 4200 ttcttgagac acagctcggg ctggatcaag tgagctactc tggttttatt gaacagctga 4260 aataaccaac tttttggaaa ttgatgaaat cttacggagt taacagtgga ggtaccaggg 4320 ctcttaagag ttcccgattc tcttctgaga ctacaaattg tgattttgca tgccacctta 4380 atcttttttt tttttttttt aaatcgaggt ttcagtctca ttctatttcc caggctggag 4440 ttcaatagcg tgatcacagc tcactgtagc cttgaactcc tggccttaag agattctcct 4500 gcttcggtct cccaatagct aagactacag tagtccacca ccatatccag ataattttta 4560 aattttttgg ggggccgggc acagtggctc acgcctgtaa tcccaacacc atgggaggct 4620 gagatgggtg gatcacgagg tcaggagttt gagaccagcc tgaccaacat ggtgaaactc 4680 tgtctctact aaaaaaaaaa aaaatagaaa aattagccgg gcgtggtggc acacggcacc 4740 tgtaatccca gctactgagg aggctgaggc aggagaatca cttgaaccca gaaggcagag 4800 gttgcaatga gccgagattg cgccactgca ctccagcctg ggtgacagag tgagactctg 4860 tctcaaaaaa aaaaaatttt tttttttttt ttgtagagat ggatcttgct ttgtttctct 4920 ggttggcctt gaactcctgg cttcaagtga tcctcctacc ttggcctcgg aaagtgttgg 4980 gattacaggc gtgagccacc atgactgacc tgtcgttaat cttgaggtac ataaacctgg 5040 ctcctaaagg ctaaaggcta aatatttgtt ggagaagggg cattggattt tgcatgagga 5100 tgattctgac ctgggagggc aggtcagcag gcatctctgt tgcacagata gagtgtacag 5160 gtctggagaa caaggagtgg ggggttattg gaattccaca ttgtttgctg cacgttggat 5220 tttgaaatgc tagggaactt tgggagactc atatttctgg gctagaggat ctgtggacca 5280 caagatcttt ttatgatgac agtagcaatg tatctgtgga gctggattct gggttgggag 5340 tgcaaggaaa agaatgtact aaatgccaag acatctattt caggagcatg aggaataaaa 5400 gttctagttt ctggtctcag agtggtgcat ggatcaggga gtctcacaat ctcctgagtg 5460 ctggtgtctt agggcacact gggtcttgga gtgcaaagga tctaggcacg tgaggctttg 5520 tatgaagaat cggggatcgt acccaccccc tgtttctgtt tcatcctggg catgtctcct 5580 ctgcctttgt cccctagatg aagtctccat gagctacaag ggcctggtgc atccagggtg 5640 atctagtaat tgcagaacag caagtgctag ctctccctcc ccttccacag ctctgggtgt 5700 gggagggggt tgtccagcct ccagcagcat ggggagggcc ttggtcagcc tctgggtgcc 5760 agcagggcag gggcggagtc ctggggaatg aaggttttat agggctcctg ggggaggctc 5820 cccagcccca agctt 5835 <210> 3 <211> 12047 <212> DNA
<213> Homo Sapiens <400> 3 gaattcagaa ataggggaag gttgaggaag gacactgaac tcaaagggga tacagtgatt 60 ggtttatttg tcttctcttc acaacattgg tgctggagga attcccaccc tgaggttatg 120 aagatgtctg aacacccaac acatagcact ggagatatga gctcgacaag agtttctcag 180 ccacagagat tcacagccta gggcaggagg acactgtacg ccaggcagaa tgacatggga 240 attgcgctca cgattggctt gaagaagcaa ggactgtggg aggtgggctt tgt,agtaaca 300 agagggcagg gtgaactctg attcccatgg gggaatgtga tggtcctgtt acaaattttt 360 caagctggca gggaataaaa cccattacgg tgaggacctg tggagggcgg ctgccccaac 420 tgataaagga aatagccagg tgggggcctt tcccattgta ggggggacat atctggcaat 480 agaagccttt gagacccttt agggtacaag tactgaggca gcaaataaaa tgaaatctta 540 tttttcaact ttatactgca tgggtgtgaa gatatatttg tttctgtaca gggggtgagg 600 gaaaggaggg gaggaggaaa gttcctgcag gtctggtttg gtcttgtgat ccagggggtc 660 ttggaactat ttaaattaaa ttaaattaaa acaagcgact gttttaaatt aaattaaatt 720 aaattaaatt ttactttatt ttatcttaag ttctgggcta catgtgcagg acgtgcagct 780 ttgttacata ggtaaacgtg tgccatggtg gtttgctgta cctatcaacc catcacctag 840 gtattaagcc cagcatgcat tagctgtttt tcctgacgct ctccctctcc ctgactccca 900 caacaggccc cagtgtgtgt tgttcccctc cctgtgtcca tgtgttctca ttgttcagct 960 cccacttata agtgagaaca tgtggtgttt ggttttctgt ttctgtgtta gtttgctgag 1020 gataatggct tccacctcca tccatgttcc tgcaaaggac gtgatcttat tcttttttat 1080 ggttgcatag aaattgtttt tacaaatcca attgatattg tatttaatta caagttaatc 1140 taattagcat actagaagag attacagaag atattaggta cattgaatga ggaaatatat 1200 aaaataggac gaaggtgaaa tattaggtag gaaaagtata atagttgaaa gaagtaaaaa 1260 aaaatatgca tgagtagcag aatgtaaaag aggtgaagaa cgtaatagtg actttttaga 1320 ccagattgaa ggacagagac agaaaaattt taaggaattg ctaaaccatg tgagtgttag 1380 aagtacagtc aataacatta aagcctcagg aggagaaaag aataggaaag gaggaaatat 1440 gtgaataaat agtagagaca tgtttgatgg attttaaaat atttgaaaga cctcacatca 1500 aaggattcat accgtgccat tgaagaggaa gatggaaaag ccaagaagcc agatgaaagt 1560 tagaaatatt attggcaaag cttaaatgtt aaaagtccta gagagaaagg atggcagaaa 1620 tattggcggg aaagaatgca gaacctagaa tataaattca tcccaacagt ttggtagtgt 1680 gcagctgtag ccttttctag ataatacact attgtcatac atcgcttaag cgagtgtaaa 1740 atggtctcct cactttattt atttatatat ttatttagtt ttgagatgga gcctcgctct 1800 gtctcctagg ctggagtgca atagtgcgat accactcact gcaacctctg cctcctctgt 1860 tcaagtgatt ttcttacctc agcctcccga gtagctggga ttacaggtgc gtgccaccac 1920 acccggctaa tttttgtatt ttttgtagag acggggtttt gccatgttgg ccaggctggt 1980 cttgaactcc tgacatcagg tgatccacct gccttggcct cctaaagtgc tgggattaca 2040 ggcatgagcc accgtgccca accactttat ttatttttta tttttatttt taaatttcag 2100 cttctatttg aaatacaggg ggcacatata taggattgtt acatgggtat attgaactca 2160 ggtagtgatc atactaccca acaggtaggt tttcaaccca ctccccctct tttcctcccc 2220 attctagtag tgtgcagtgt ctattgttct catgtttatg tctatgtgtg ctccaggttt 2280 agctcccacc tgtaagtgag aacgtgtggt atttgatttt ctgtccctgt gttaattcac 2340 ttaggattat ggcttccagc tccattcata ttgctgtaaa ggatatgatt catttttcat 2400 ggccatgcag tattccatat tgcgtataga tcacattttc tttctttttt ttttttgaga 2460 cggagtcttg ctttgctgcc taggctggag tgcagtagca cgatctcggc tcactgcaag 2520 cttcacctcc ggggttcacg tcattcttct gtctcagctt cccaagtagc tgggactaca 2580 ggcgcccgcc accacgtccg gctaattttt ttgtgtgttt ttagtagaga tgggggtttc 2640 actgtgttag ccaggatggt cttgatctcc tgaccttgtg gtccacctgc ctcggtctcc 2700 caaagtgctg ggattacagg ggtgagccac tgcgcccggc ccatatatac cacattttct 2760 ttaaccaatc caccattgat gggcaactag gtagattcca tggattccac agttttgcta 2820 ttgtgtgcag tgtggcagta gacatatgaa tgaatgtgtc tttttggtat aatgatttgc 2880 attcctttgg gtatacagtc attaatagga gtgctgggtt gaacggtggc tctgtttaaa 2940 attctttgag aattttccaa actgtttgcc atagagagca aactaattta catttccacg 3000 aacagtatat aagcattccc ttttctccac agctttgtca tcatggtttt tttttttctt 3060 tattttaaaa aagaatatgt tgttgttttc ccagggtaca tgtgcaggat gtgcaggttt 3120 gttacatagg tagtaaacgt gagccatggt ggtttgctgc acctgtcaac ccattacctg 3180 ggtatgaagc cctgcctgca ttagctcttt tccctaatgc tctcactact gccccaccct 3240 caccctgaca gggcaaacag acaacctaca gaatgggagg aaatttttgc aatctattca 3300 tctgacaaag gtcaagaata tccagaatct acaaggaact taagcaaatt tttacttttt 3360 aataatagcc actctgactg gcgtgaaatg gtatctcatt gtggttttca tttgaatttc 3420 tctgatgatc agtgacgatg agcatttttt catatttgtt ggctgcttgt acgtcttttg 3480 agaagtgtct cttcatgcct tttggccact ttaatgggat tattttttgc tttttagttt 3540 aagttcctta tagattctgg atattagact tcttattgga tgcatagttt gtgaatactc 3600 tcttccattc tgtaggttgt ctgtttactc tattgatggc ttcttttgct gtgccgaagc 3660 atcttagttt aattagaaac cacctgccaa tttttgtttt tgttgcaatt gcttttgggg 3720 acttagtcat aaactctttg ccaaggtctg ggtcaagaag agtatttcct aggttttctt 3780 ctagaatttt gaaagtctga atgtaaacat ttgcattttt aatgcatctt gagttagttt 3840 ttgtatatgt gaaaggtcta ctctcatttt ctttccctct ttctttcttt ctttcttttc 3900 tttctttctt tctttctttc tttctttctt tctttctttc tttctttttg tccttctttc 3960 tttctttctt tctctttctt tctctctttc tttttttttt ttgatggagt attgctctgt 4020 tgcccaggct gcagtgcagc ggcacgatct cggctcactg caacctctgc ctcctgggtt 4080 caactgattc tcctgcatca gccttccaag tagctgggat tataggcgcc cgccaccacg 4140 cccgactaat ttttgtattt ttagtagaga cggggttgtg ccatgttggc caggctggtt 4200 tgaaactcct gacctcaaac gatctgcctg ccttggcctc ccaaagtgct gggattacag 4260 gtgtgagcca ctgtgcccag ccaagaatgt cattttctaa gaggtccaag aacctcaaga 4320 tattttggga ccttgagaag agaggaattc atacaggtat tacaagcaca gcctaatggc 4380 aaatctttgg catggcttgg cttcaagact ttaggctctt aaaagtcgaa tccaaaaatt 4440 tttataaaag ctccagctaa gctaccttaa aaggggcctg tatggctgat cactcttctt 4500 gctatacttt acacaaataa acaggccaaa tataatgagg ccaaaattta ttttgcaaat 4560 aaattggtcc tgctatgatt tactcttggt aagaacaggg aaaatagaga aaaatttaga 4620 ttgcatctga cctttttttc tgaattttta tatgtgccta caatttgagc taaatcctga 4680 attattttct ggttgcaaaa actctctaaa gaagaacttg gttttcattg tcttcgtgac 4740 acatttatct ggctctttac tagaacagct ttcttgtttt tggtgttcta gcttgtgtgc 4800 cttacagttc tactcttcaa attattgtta tgtgtatctc atagttttcc ttcttttgag 4860 aaaactgaag ccatggtatt ctgaggacta gagatgactc aacagagctg gtgaatctcc 4920 tcatatgcaa tccactgggc tcgatctgct tcaaattgct gatgcactgc tgctaaagct 4980 atacatttaa aaccctcact aaaggatcag ggaccatcat ggaagaggag gaaacatgaa 5040 attgtaagag ccagattcgg ggggtagagt gtggaggtca gagcaactcc accttgaata 5100 agaaggtaaa gcaacctatc ctgaaagcta acctgccatg gtggcttctg attaacctct 5160 gttctaggaa gactgacagt ttgggtctgt gtcattgccc aaatctcatg ttaaattgta 5220 atccccagtg ttcggaggtg ggacttggtg gtaggtgatt cggtcatggg agtagatttt 5280 cttctttgtg gtgttacagt gatagtgagt gagttctcgt gagatctggt catttaaaag 5340 tgtgtggccc ctcccctccc tctcttggtc ctcctactgc catgtaagat acctgctcct 5400 gctttgcctt ctaccataag taaaagcccc ctgaggcctc cccagaagca gatgccacca 5460 tgcttcctgt acagcctgca gaaccatcag ccaattaaac ctcttttctg tataaattac 5520 cagtcttgag tatctcttta cagcagtgtg agaacggact aatacaaggg tctccaaaat 5580 tccaagttta tgtattcttt cttgccaaat agcaggtatt taccataaat cctgtcctta 5640 ggtcaaacaa ccttgatggc atcgtacttc aattgtctta cacattcctt ctgaatgact 5700 cctcccctat ggcatataag ccctgggtct tgggggataa tggcagaggg gtccaccatc 5760 ttgtctggct gccacctgag acacggacat ggcttctgtt ggtaagtctc tattaaatgt 5820 ttctttctaa gaaactggat ttgtcagctt gtttctttgg cctctcagct tcctcagact 5880 ttggggtagg ttgcacaacc ctgcccacca cgaaacaaat gtttaatatg ataaatatgg 5940 atagatataa tccacataaa taaaagctct tggagggccc tcaataattg ttaagagtgt 6000 aaatgtgtcc aaagatggaa aatgtttgag aactactgtc ccagagattt tcctgagttc 6060 tagagtgtgg gaatatagaa cctggagctt ggcttcttca gcctagaatc aggagtatgg 6120 ggctgaagtc tgaagcttgg cttcagcagt ttggggttgg cttccggagc acatatttga 6180 catgttgcga ctgtgatttg gggtttggta tttgctctga atcctaatgt ctgtccttga 6240 ggcatctaga atctgaaatc tgtggtcaga attctattat cttgagtagg acatctccag 6300 tcctggttct gccttctagg gctggagtct gtagtcagtg acccggtctg gcatttcaac 6360 ttcatataca gtgggctatc ttttggtcca tgtttcaacc aaacaaccga ataaaccatt 6420 agaacctttc cccacttccc tagctgcaat gttaaaccta ggatttctgt ttaataggtt 6480 catatgaata atttcagcct gatccaactt tacattcctt ctaccgttat tctacaccca 6540 ccttaaaaat gcattcccaa tatattccct ggattctacc tatatatggt aatcctggct 6600 ttgccagttt ctagtgcatt aacatacctg atttacattc ttttacttta aagtggaaat 6660 aagagtccct ctgcagagtt caggagttct caagatggcc cttacttctg acatcaattg 6720 agatttcaag ggagtcgcca agatcatcct caggttcagt gattgctggt agccctcata 6780 taactcaatg aaagctgtta tgctcatggc tatggtttat tacagcaaaa gaatagagat 6840 gaaaatctag caagggaaga gttgcatggg gcaaagacaa ggagagctcc aagtgcagag 6900 attcctgttg ttttctccca gtggtgtcat ggaaagcagt atcttctcca tacaatgatg 6960 tgtgataata ttcagtgtat tgccaatcag ggaactcaac tgagccttga ttatattgga 7020 gcttggttgc acagacatgt cgaccacctt catggctgaa ctttagtact tagcccctcc 7080 agacgtctac agctgatagg ctgtaaccca acattgtcac cataaatcac attgttagac 7140 tatccagtgt ggcccaagct cccgtgtaaa cacaggcact ctaaacaggc aggatatttc 7200 aaaagcttag agatgacctc ccaggagctg aatgcaaaga cctggcctct ttgggcaagg 7260 agaatccttt accgcacact ctccttcaca gggttattgt gaggatcaaa tgtggtcatg 7320 tgtgtgagac accagcacat gtctggctgt ggagagtgac ttctatgtgt gctaacattg 7380 ctgagtgcta agaaagtatt aggcatggct ttcagcactc acagatgctc atctaatcct 7440 cacaacatgg ctacagggtg ggcactacta gcctcatttg acagaggaaa ggactgtgga 7500 taagaagggg gtgaccaata ggtcagagtc attctggatg caaggggctc cagaggacca 7560 tgattagaca ttgtctgcag agaaattatg gctggatgtc tctgccccgg aaagggggat 7620 gcactttcct tgacccccta tctcagatct tgactttgag gttatctcag acttcctcta 7680 tgataccagg agcccatcat aatctctctg tgtcctctcc ccttcctcag tcttactgcc 7740 cactcttccc agctccatct ccagctggc,c aggtgtagcc acagtaccta actctttgca 7800 gagaactata aatgtgtatc ctacagggga gaaaaaaaaa aagaactctg aaagagctga 7860 cattttaccg acttgcaaac acataagcta acctgccagt tttgtgctgg tagaactcat 7920 gagactcctg ggtcagaggc aaaagatttt attacccaca gctaaggagg cagcatgaac 7980 tttgtgttca catttgttca ctttgccccc caattcatat gggatgatca gagcagttca 8040 ggtggatgga cacaggggtt tgtggcaaag gtgagcaacc taggcttaga aatcctcaat 8100 cttataagaa ggtactagca aacttgtcca gtctttgtat ctgacggaga tattatcttt 8160 ataattgggt tgaaagcaga cctactctgg aggaacatat tgtatttatt gtcctgaaca 8220 gtaaacaaat ctgctgtaaa atagacgtta actttattat ctaaggcagt aagcaaacct 8280 agatctgaag gcgataccat cttgcaaggc tatctgctgt acaaatatgc ttgaaaagat 8340 ggtccagaaa agaaaacggt attattgcct ttgctcagaa gacacacaga aacataagag 8400 aaccatggaa aattgtctcc caacactgtt cacccagagc cttccactct tgtctgcagg 8460 acagtcttaa catcccatca ttagtgtgtc taccacatct ggcttcaccg tgcctaacca 8520 agatttctag gtccagttcc ccaccatgtt tggcagtgcc ccactgccaa ccccagaata 8580 agggagtgct cagaattccg aggggacatg ggtggggatc agaacttctg ggcttgagtg 8640 cagagggggc ccatactcct tggttccgaa ggaggaagag gctggaggtg aatgtccttg 8700 gaggggagga atgtgggttc tgaactctta aatccccaag ggaggagact ggtaaggtcc 8760 cagcttccga ggtactgacg tgggaatggc ctgagaggtc taagaatccc gtatcctcgg 8820 gaaggagggg ctgaaattgt gaggggttga gttgcagggg tttgttagct tgagactcct 8880 tggtgggtcc ctgggaagca aggactggaa ccattggctc cagggtttgg tgtgaaggta 8940 atgggatctc ctgattctca aagggtcaga ggactgagag ttgcccatgc tttgatcttt 9000 ccatctactc cttactccac ttgagggtaa tcacctactc ttctagttcc acaagagtgc 9060 gcctgcgcga gtataatctg cacatgtgcc atgtcccgag gcctggggca tcatccactc 9120 atcattcagc atctgcgcta tgcgggcgag gccggcgcca tgacgtcatg tagctgcgac 9180 tatccctgca gcgcgcctct cccgtcacgt cccaaccatg gagctgtgga cgtgcgtccc 9240 ctggtggatg tggcctgcgt ggtgccaggc cggggcctgg tgtccgataa agatcctaga 9300 accacaggaa accaggactg aaaggtgcta gagaatggcc atatgtcgct gtccatgaaa 9360 tctcaaggac ttctgggtgg agggcacagg agcctgaact tacgggtttg ccccagtcca 9420 ctgtcctccc aagtgagtct cccagatacg aggcactgtg ccagcatcag cttcatctgt 9480 accacatctt gtaacaggga ctacccagga ccctgatgaa caccatggtg tgtgcaggaa 9540 gagggggtga aggcatggac tcctgtgtgg tcagagccca gagggggcca tgacgggtgg 9600 ggaggaggct gtggactggc tcgagaagtg ggatgtggtt gtgtttgatt tcctttggcc 9660 agataaagtg ctggatatag cattgaaaac ggagtatgaa gaccagttag aatggagggt 9720 caggttggag ttgagttaca gatggggtaa aattctgctt cggatgagtt tggggattgg 9780 caatctaaag gtggtttggg atggcatggc tttgggatgg aaataggttt gtttttatgt 9840 tggctgggaa gggtgtgggg attgaattgg ggatgaagta ggtttagttt tggagataga 9900 atacatggag ctggctattg catgcgagga tgtgcattag tttggtttga tctttaaata 9960 aaggaggcta ttagggttgt cttgaattag attaagttgt gttgggttga tgggttgggc 10020 ttgtgggtga tgtggttgga ttgggctgtg ttaaattggt ttgggtcagg ttttggttga 10080 ggttatcatg gggatgagga tatgcttggg acatggattc aggtggttct cattcaagct 10140 gaggcaaatt tcctttcaga cggtcattcc agggaacgag tggttgtgtg ggggaaatca 10200 ggccactggc tgtgaatatc cctctatcct ggtcttgaat tgtgattatc tatgtccatt 10260 ctgtctcctt cactgtactt ggaattgatc tggtcattca gctggaaatg ggggaagatt 10320 ttgtcaaatt cttgagacac agctgggtct ggatcagcgt aagccttcct tctggtttta 10380 ttgaacagat gaaatcacat tttttttttc aaaatcacag aaatcttata gagttaacag 10440 tggactctta taataagagt taacaccagg actcttattc ttgattcttt tctgagacac 10500 caaaatgaga tttctcaatg ccaccctaat tctttttttt tttttttttt tttttgagac 10560 acagtctggg tcttttgctc tgtcactcag gctggagcgc agtggtgtga tcatagctca 10620 ctgaaccctt gacctcctgg acttaaggga tcctcctgct tcagcctcct gagtagatgg 10680 ggctacaggt gcttgccacc acacctggct aattaaattt tttttttttt tttgtagaga 10740 aagggtctca ctttgttgcc ctggctgatc ttgaacttct gacttcaagt gattcttcag 10800 ccttggactc ccaaagcact gggattgctg gcatgagcca ctcaccgtgc ctggcttgca 10860 gcttaatctt ggagtgtata aacctggctc ctgatagcta gacatttcag tgagaaggag 10920 gcattggatt ttgcatgagg acaattctga cctaggaggg caggtcaaca ggaatccccg 10980 ctgtacctgt acgttgtaca ggcatggaga atgaggagtg aggaggccgt accggaaccc 11040 catattgttt agtggacatt ggattttgaa ataataggga acttggtctg ggagagtcat 11100 atttctggat tggacaatat gtggtatcac aaggttttat gatgagggag aaatgtatgt 11160 ggggaaccat tttctgagtg tggaagtgca agaatcagag agtagctgaa tgccaacgct 11220 tctatttcag gaacatggta agttggaggt ccagctctcg ggctcagacg ggtataggga 11280 ccaggaagtc tcacaatccg atcattctga tatttcaggg catattaggt ttggggtgca 11340 aaggaagtac ttgggactta ggcacatgag actttgtatt gaaaatcaat gattggggct 11400 ggccgtggtg ctcacgcctg taatctcatc actttgggag accgaagtgg gaggatggct 11460 tgatctcaag agttggacac cagcctaggc aacatggcca gaccctctct ctacaaaaaa 11520 attaaaaatt agctggatgt ggtggtgcat gcttgtggtc tcagctatcc tggaggctga 11580 gacaggagaa tcggttgagt ctgggagttc aaggctacag ggagctgcga tcacgccgct 11640 gcactccagc ctgggaaaca gagtgagact gtctcagaat ttttttaaaa aagaatcagt 11700 gatcatccca acccctgttg ctgttcatcc tgagcctgcc ttctctggct ttgttcccta 11760 gatcacatct ccatgatcca taggccctgc ccaatctgac ctcacaccgt gggaatgcct 11820 ccagactgat ctagtatgtg tggaacagca agtgctggct ctccctcccc ttccacagct 11880 ctgggtgtgg gagggggttg tccagcctcc agcagcatgg ggagggcctt ggtcagcatc 11940 taggtgccaa cagggcaagg gcggggtcct ggagaatgaa ggctttatag ggctcctcag 12000 ggaggccccc cagccccaaa ctgcaccacc tggccgtgga caccggt 12047 <210> 4 <211> 454 <212> DNA
<213> Homo Sapiens <400> 4 aagcttccac aagtgcattt agcctctcca gtattgctga tgaatccaca gttcaggttc 60 aatggcgttc aaaacttgat caaaaatgac cagactttat attcttacac caacatctat 120 ctgattggag gaatggataa tagtcatcat gtttaaacat ctaccattcc agttaagaaa 180 atatgatagc atcttgttct tagtcttttt cttaataggg acataaagcc cacaaataaa 240 aatatgcctg aagaatggga caggcattgg gcattgtcca tgcctagtaa agtactccaa 300 gaacctattt gtatactaga tgacacaatg tcaatgtctg tgtacaactg ccaactggga 360 tgcaagacac tgcccatgcc aatcatcctg aaaagcagct ataaaaagca ggaagctact 420 ctgcaccttg tcagtgaggt ccagatacct acag 454 <210> 5 <211> 5224 <212> DNA
<213> Homo Sapiens <400> 5 gaattcttag aaatatgggg gtaggggtgg tggtggtaat tctgttttca ccccataggt 60 gagataagca ttgggttaaa tgtgctttca cacacacatc acatttcata agaattaagg 120 aacagactat gggctggagg actttgagga tgtctgtctc ataacacttg ggttgtatct 180 gttctatggg gcttgtttta agcttggcaa cttgcaacag ggttcactga ctttctcccc 240 aagcccaagg tactgtcctc ttttcatatc tgttttgggg cctctggggc ttgaatatct 300 gagaaaatat aaacatttca ataatgttct gtggtgagat gagtatgaga gatgtgtcat 360 tcatttgtat caatgaatga atgaggacaa ttagtgtata aatccttagt acaacaatct 420 gagggtaggg gtggtactat tcaatttcta tttataaaga tacttatttc tatttattta 480 tgcttgtgac aaatgttttg ttcgggacca caggaatcac aaagatgagt ctttgaattt 540 aagaagttaa tggtccagga ataattacat agcttacaaa tgactatgat ataccatcaa 600 acaagaggtt ccatgagaaa ataatctgaa aggtttaata agttgtcaaa ggtgagaggg 660 ctcttctcta gctagagact aatcagaaat acattcaggg ataattattt gaatagacct 720 taagggttgg gtacattttg ttcaagcatt gatggagaag gagagtgaat atttgaaaac 780 attttcaact aaccaaccac ccaatccaac aaacaaaaaa tgaaaagaat ctcagaaaca 840 gtgagataag agaaggaatt ttctcacaac ccacacgtat agctcaactg ctctgaagaa 900 gtatatatct aatatttaac actaacatca tgctaataat gataataatt actgtcattt 960 tttaatgtct ataagtacca ggcatttaga agatattatt ccatttatat atcaaaataa 1020 acttgagggg atagatcatt ttcatgatat atgagaaaaa ttaaaaacag attgaattat 1080 ttgcctgtca tacagctaat aattgaccat aagacaatta gatttaaatt agttttgaat 1140 ctttctaata ccaaagttca gtttactgtt ccatgttgct tctgagtggc ttcacagact 1200 tatgaaaaag taaacggaat cagaattaca tcaatgcaaa agcattgctg tgaactctgt 1260 acttaggact aaactttgag caataacaca catagattga ggattgtttg ctgttagcat 1320 acaaactctg gttcaaagct cctctttatt gcttgtcttg gaaaatttgc tgttcttcat 1380 ggtttctctt ttcactgcta tctatttttc tcaaccactc acatggctac aataactgtc 1440 tgcaagctta tgattcccaa atatctatct ctagcctcaa tcttgttcca gaagataaaa 1500 agtagtattc aaatgcacat caacgtctcc acttggaggg cttaaagacg tttcaacata 1560 caaaccgggg agttttgcct ggaatgtttc ctaaaatgtg tcctgtagca catagggtcc 1620 tcttgttcct taaaatctaa ttacttttag cccagtgctc atcccaccta tggggagatg 1680 agagtgaaaa gggagcctga ttaataatta cactaagtca ataggcatag agccaggact 1740 gtttgggtaa actggtcact ttatcttaaa ctaaatatat ccaaaactga acatgtactt 1800 agttactaag tctttgactt tatctcattc ataccactca gctttatcca ggccacttat 1860 ttgacagtat tattgcgaaa acttcctaac tggtctcctt atcatagtct tatccccttt 1920 tgaaacaaaa gagacagttt caaaatacaa atatgatttt tattagctcc cttttgttgt 1980 ctataatagt cccagaagga gttataaact ccatttaaaa agtctttgag atgtggccct 2040 tgccaacttt gccaggaatt cccaatatct agtattttct actattaaac tttgtgcctc 2100 ttcaaaactg cattttctct cattccctaa gtgtgcattg ttttccctta ccggttggtt 2160 tttccaccac cttttacatt ttcctggaac actataccct ccctcttcat ttggcccacc 2220 tctaattttc tttcagatct ccatgaagat gttacttcct ccaggaagcc ttatctgacc 2280 cctccaaaga tgtcatgagt tcctcttttc attctactaa tcacagcatc catcacacca 2340 tgttgtgatt actgatacta ttgtctgttt ctctgattag gcagtaagct caacaagagc 2400 tacatggtgc ctgtctcttg ttgctgatta ttcccatcca aaaacagtgc ctggaatgca 2460 gacttaacat tttattgaat gaataaataa aaccccatct atcgagtgct actttgtgca 2520 agacccggtt ctgaggcatt tatatttatt gatttattta attctcattt aaccatgaag 2580 gaggtactat cactatcctt attttatagt tgataaagat aaagcccaga gaaatgaatt 2640 aactcaccca aagtcatgta gctaagtgac agggcaaaaa ttcaaaccag ttccccaact 2700 ttacgtgatt aatactgtgc tatactgcct ctctgatcat atggcatgga atgcagacat 2760 ctgctccgta aggcagaata tggaaggaga ttggaggatg acacaaaacc agcataatat 2820 cagaggaaaa gtccaaacag gacctgaact gatagaaaag ttgttactcc tggtgtagtc 2880 gcatcgacat cttgatgaac tggtggctga cacaacatac attggcttga tgtgtacata 2940 ttatttgtag ttgtgtgtgt atttttatat atatatttgt aatattgaaa tagtcataat 3000 ttactaaagg cctaccattt gccaggcatt tttacatttg tcccctctaa tcttttgatg 3060 agatgatcag attggattac ttggccttga agatgatata tctacatcta tatctatatc 3120 tatatctata tctatatcta tatctatatc tatatctata tatgtatatc agaaaagctg 3180 aaatatgttt tgtaaagtta taaagattt~ agactttata gaatctggga tttgccaaat 3240 gtaacccctt tctctacatt aaacccatgt tggaacaaat acatttatta ttcattcatc 3300 aaatgttgct gagtcctggc tatgaaccag acactgtgaa agcctttggg atattttgcc 3360 catgcttggg caagcttata tagtttgctt cataaaactc tatttcagtt cttcataact 3420 aatacttcat gactattgct tttcaggtat tccttcataa caaatacttt ggctttcata 3480 tatttgagta aagtccccct tgaggaagag tagaagaact gcactttgta aatactatcc 3540 tggaatccaa acggatagac aaggatggtg ctacctcttt ctggagagta cgtgagcaag 3600 gcctgttttg ttaacatgtt ccttaggaga caaaacttag gagagacacg catagcagaa 3660 aatggacaaa aactaacaaa tgaatgggaa ttgtacttga ttagcattga agaccttgtt 3720 tatactatga taaatgtttg tatttgctgg aagtgctact gacggtaaac cctttttgtt 3780 taaatgtgtg ccctagtagc ttgcagtatg atctattttt taagtactgt acttagctta 3840 tttaaaaatt ttatgtttaa aattgcatag tgctctttca ttgaagaagt tttgagagag 3900 agatagaatt aaattcactt atcttaccat ctagagaaac ccaatgttaa aactttgttg 3960 tccattattt ctgtctttta ttcaacattt tttttagagg gtgggaggaa tacagaggag 4020 gtacaatgat acacaaatga gagcactctc catgtattgt tttgtcctgt ttttcagtta 4080 acaatatatt atgagcatat ttccatttca ttaaatattc ttccacaaag ttattttgat 4140 ggctgtatat caccctactt tatgaatgta ccatattaat ttatttcctg gtgtgggtta 4200 tttgatttta taatcttacc tttagaataa tgaaacacct gtgaagcttt agaaaatact 4260 ggtgcctggg tctcaactcc acagattctg atttaactgg tctgggttac agactaggca 4320 ttgggaattc aaaaagttcc cccagtgatt ctaatgtgta gccaagatcg ggaacccttg 4380 tagacaggga tgataggagg tgagccactc ttagcatcca tcatttagta ttaacatcat 4440 catcttgagt tgctaagtga atgatgcacc tgacccactt tataaagaca catgtgcaaa 4500 taaaattatt ataggacttg gtttattagg gcttgtgctc taagttttct atgttaagcc 4560 atacatcgca tactaaatac tttaaaatgt accttattga catacatatt aagtgaaaag 4620 tgtttctgag ctaaacaatg acagcataat tatcaagcaa tgataatttg aaatgaattt 4680 attattctgc aacttaggga caagtcatct ctctgaattt tttgtacttt gagagtattt 4740 gttatatttg caagatgaag agtctgaatt ggtcagacaa tgtcttgtgt gcctggcata 4800 tgataggcat ttaatagttt taaagaatta atgtatttag atgaattgca taccaaatct 4860 gctgtctttt ctttatggct tcattaactt aatttgagag aaattaatta ttctgcaact 4920 tagggacaag tcatgtcttt gaatattctg tagtttgagg agaatatttg ttatatttgc 4980 aaaataaaat aagtttgcaa gttttttttt tctgccccaa agagctctgt gtccttgaac 5040 ataaaataca aataaccgct atgctgttaa ttattggcaa atgtcccatt ttcaacctaa 5100 ggaaatacca taaagtaaca gatataccaa caaaaggtta ctagttaaca ggcattgcct 5160 gaaaagagta taaaagaatt tcagcatgat tttccatatt gtgcttccac cactgccaat 5220 aaca <210> 6 <211> 822 <212> DNA
<213> Homo Sapiens <400> 6 gcattgctgt gaactctgta cttaggacta aactttgagc aataacacac atagattgag 60 gattgtttgc tgttagcata caaactctgg ttcaaagctc ctctttattg cttgtcttgg 120 aaaatttgct gttcttcatg gtttctcttt tcactgctat ctatttttct caaccactca 180 catggctaca ataactgtct gcaagcttat gattcccaaa tatctatctc tagcctcaat 240 cttgttccag aagataaaaa gtagtattca aatgcacatc aacgtctcca cttggagggc 300 ttaaagacgt ttcaacatac aaaccgggga gttttgcctg gaatgtttcc taaaatgtgt 360 cctgtagcac atagggtcct cttgttcctt aaaatctaat tacttttagc ccagtgctca 420 tcccacctat ggggagatga gagtgaaaag ggagcctgat taataattac actaagtcaa 480 taggcataga gccaggactg tttgggtaaa ctggtcactt tatcttaaac taaatatatc 540 caaaactgaa catgtactta gttactaagt ctttgacttt atctcattca taccactcag 600 ctttatccag gccacttatg agctctgtgt ccttgaacat aaaatacaaa taaccgctat 660 gctgttaatt attggcaaat gtcccatttt caacctaagg aaataccata aagtaacaga 720 tataccaaca aaaggttact agttaacagg cattgcctga aaagagtata aaagaatttc 780 agcatgattt tccatattgt gcttccacca ctgccaataa ca 822 <210> 7 <211> 472 <212> DNA
<213> Homo Sapiens <400> 7 agccaccacc cagtgagcct ttttctagcc cccagagcca cctctgtcac cttcctgttg 60 ggcatcatcc caccttccca gagccctgga gagcatgggg agacccggga ccctgctggg 120 tttctctgtc acaaaggaaa ataatccccc tggtgtgaca gacccaagga cagaacacag 180 cagaggtcag cactggggaa gacaggttgt cctcccaggg gatgggggtc catccacctt 240 gccgaaaaga tttgtctgag gaactgaaaa tagaagggaa aaaagaggag ggacaaaaga 300 ggcagaaatg agaggggagg ggacagagga cacctgaata aagaccacac ccatgaccca 360 cgtgatgctg agaagtactc ctgccctagg aagagactca gggcagaggg aggaaggaca 420 gcagaccaga cagtcacagc agccttgaca aaacgttcct ggaactcaag ca 472 <210> 8 <211> 858 <212> DNA
<213> Homo Sapiens <400> 8 cgagcggccc ctcagcttcg gcgcccagcc ccgcaaggct cccggtgacc actagagggc 60 gggaggagct cctggccagt ggtggagagt ggcaaggaag gaccctaggg ttcatcggag 120 cccaggttta ctcccttaag tggaaatttc ttcccccact cctccttggc tttctccaag 180 gaggga~ccc aggctgctgg aaagtccggc tggggcgggg actgtgggtt caggggagaa 240 cggggtgtgg aacgggacag ggagcggtta gaagggtggg gctattccgg gaagtggtgg 300 ggggagggag cccaaaacta gcacctagtc cactcattat ccagccctct tatttctcgg 360 ccgctctgct tcagtggacc cggggagggc ggggaagtgg agtgggagac ctaggggtgg 420 gcttcccgac cttgctgtac aggacctcga cctagctggc tttgttcccc atccccacgt 480 tagttgttgc cctgaggcta aaactagagc ccaggggccc caagttccag actgcccctc 540 ccccctcccc cggagccagg gagtggttgg tgaaaggggg aggccagctg gagaacaaac 600 gggtagtcag ggggttgagc gattagagcc cttgtaccct acccaggaat ggttggggag 660 gaggaggaag aggtaggagg taggggaggg ggcggggttt tgtcacctgt cacctgctcg 720 ctgtgcctag ggcgggcggg cggggagtgg ggggaccggt ataaagcggt aggcgcctgt 780 gcccgctcca cctctcaagc agccagcgcc tgcctgaatc tgttctgccc cctccccacc 840 catttcacca ccaccatg 858 <210> 9 <211> 454 <212> DNA
<213> Homo Sapiens <400> 9 aagcttccac aagtgcattt agcctctcca gtattgctga tgaatccaca gttcaggttc 60 aatggcgttc aaaacttgat caaaaatgac cagactttat attcttacac caacatctat 120 ctgattggag gaatggataa tagtcatcat gtttaaacat ctaccattcc agttaagaaa 180 atatgatagc atcttgttct tagtcttttt cttaataggg acataaagcc cacaaataaa 240 aatatgcctg aagaatggga caggcattgg gcattgtcca tgcctagtaa agtactccaa 300 gaacctattt gtatactaga tgacacaatg tcaatgtctg tgtacaactg ccaactggga 360 tgcaagacac tgcccatgcc aatcatcctg aaaagcagct ataaaaagca ggaagctact 420 ctgcaccttg tcagtgaggt ccagatacct acag 454 adenovirus death protein <210> 10 <211> 307 <212> DNA
<213> adenovirus <220>
<221> CDS

<222> (2)..(304) <400> 10 g atg acc ggc tca acc atc gcg ccc aca acg gac tat cgc aac acc act 49 Met Thr Gly Ser Thr Ile Ala Pro Thr Thr Asp Tyr Arg Asn Thr Thr get acc gga cta aca tct gcc cta aat tta ccc caa gtt cat gcc ttt 97 Ala Thr Gly Leu Thr Ser Ala Leu Asn Leu Pro G1n Val His Ala Phe gtc aat gac tgg gcg agc ttg gac atg tgg tgg ttt tcc ata gcg ctt 145 Val Asn Asp Trp Ala Ser Leu Asp Met Trp Trp Phe Ser Ile Ala Leu atg ttt gtt tgc ctt att att atg tgg ctt att tgt tgc cta aag cgc 193 Met Phe Val Cys Leu Ile Ile Met Trp Leu Ile Cys Cys Leu Lys Arg aga cgc gcc aga ccc ccc atc tat agg cct atc att gtg ctc aac cca 241 Arg Arg Ala Arg Pro Pro Ile Tyr Arg Pro Ile Ile Val Leu Asn Pro cac aat gaa aaa att cat aga ttg gac ggt ctg aaa cca tgt tct ctt 289 His Asn Glu Lys Ile His Arg Leu Asp Gly Leu Lys Pro Cys Ser Leu ctt tta cag tat gat taa 307 Leu Leu Gln Tyr Asp <210> 11 <211> 101 <212> PRT
<213> adenovirus <400> 11 Met Thr Gly Ser Thr Ile Ala Pro Thr Thr Asp Tyr Arg Asn Thr Thr Ala Thr Gly Leu Thr Ser Ala Leu Asn Leu Pro Gln Val His Ala Phe Val Asn Asp Trp Ala Ser Leu Asp Met Trp Trp Phe Ser Ile Ala Leu Met Phe Va1 Cys Leu 21e Ile Met Trp Leu Ile Cys Cys Leu Lys Arg Arg Arg Ala Arg Pro Pro 21e Tyr Arg Pro Ile Ile Val Leu Asn Pro His Asn G1u Lys Ile His Arg Leu Asp Gly Leu Lys Pro Cys Ser Leu Leu Leu Gln Tyr Asp

Claims (27)

1. A replication competent adenovirus vector comprising:
an inducible transcriptional transactivator coding sequence under the transcriptional control of a cell type-specific transcriptional response element (CT-TRE);
an adenovirus gene under transcriptional control of a transcriptional response element regulated by said transcriptional transactivator;
wherein said transcriptional transactivator is functionally responsive to an exogenous inducing agent.

2. The adenovirus vector according to claim 1, wherein the adenovirus gene is a gene essential for adenoviral replication.

3. The adenovirus vector according to claim 2, wherein the gene essential for replication is an adenoviral early gene.

4. The adenovirus vector according to claim 3, wherein the adenovirus early gene is E1A.

5. The adenovirus vector according to claim 3, wherein the adenovirus early gene is E1B.

6. The adenovirus vector of claim 2, wherein the gene essential for adenoviral replication is the adenovirus E4 gene.

7. The adenovirus vector of claim 2, wherein the gene essential for adenoviral replication is an adenovirus late gene.

8. The adenovirus vector of Claim 1, wherein said transcriptional transactivator is inhibited by said inducing agent.

9. The adenovirus of Claim 1, wherein said transcriptional transactivator is activated by said inducing agent.

10. The adenovirus of Claim 1, wherein said inducing agent is selected from the group consisting of tetracycline, doxycycline and analogs thereof, FKBP
homodimers and heterodumers, ecdysone, rapamycin and analogs thereof.

11. An adenovirus vector comprising:
an inducible transcriptional transactivator coding sequence under the transcriptional control of a cell type-specific transcriptional response element (CT-TRE);
an adenovirus gene under transcriptional control of a transcriptional response element regulated by said transcriptional transactivator and a second gene under transcriptional control of a second transcriptional response element;
wherein said transcriptional transactivator is activated by an exogenous inducing agent.

12. The adenovirus vector according to claim 11, wherein the second gene is an adenoviral gene.

13. The adenovirus vector according to claim 12, wherein the adenoviral gene is a gene essential for adenoviral replication.

14. The adenovirus vector according to claim 13, wherein the gene essential for adenoviral replication is an adenovirus early gene.

15. The adenovirus vector according to claim 14, wherein the adenovirus early gene is E1A.

16. The adenovirus vector according to claim 14, wherein the adenovirus early gene is E1B.

17. The adenovirus vector of claim 12, wherein the first and second genes are essential for adenovirus replication.

18. The adenovirus vector of claim 17, wherein the first and second genes are adenovirus early genes.

19. The adenovirus vector of claim 18, wherein the first gene is E1A and the second gene E1B.

20. The adenovirus vector of claim 17, wherein the expression of the first second genes is regulated by an IRES.

21. The adenovirus vector of claim 11, wherein the second gene is a transgene.

22. The adenovirus vector of claim 21, wherein said transgene is a cytotoxic gene.

23. A isolated host cell comprising the adenovirus vector of claim 1.

24. A composition comprising the adenovirus vector of claim 1 and a pharmaceutically acceptable excipient.

25. A method for propagating a replication competent adenovirus vector comprising an inducible transcriptional transactivator coding sequence under the transcriptional control of a cell type-specific transcriptional response element (CT-TRE); an adenovirus gene under transcriptional control of a transcriptional response element regulated by said transcriptional transactivator; wherein said transcriptional transactivator is activated by an exogenous inducing agent, the method comprising:
introducing said adenovirus vector into a cell which allows the cell-specific TRE to function;
administering said inducing agent;
wherein said adenovirus vector is propagated.

26. A method for regulating a replication competent adenovirus vector comprising an inducible transcriptional transactivator coding sequence under the transcriptional control of a cell type-specific transcriptional response element (CT-TRE); an adenovirus gene under transcriptional control of a transcriptional response element regulated by said transcriptional transactivator; wherein said transcriptional transactivator is inhibited by an exogenous inducing agent, the method comprising:
introducing said adenovirus vector into a cell which allows the cell-specific THE to function wherein said adenovirus vector is propagated;
administering said inducing agent, wherein propagation of said adenovirus is inhibited.

27. A method for selective cytolysis of a target tumor cell the method comprising:
an inducible transcriptional transactivator coding sequence under the transcriptional control of a cell type-specific transcriptional response element (CT-TRE); an adenovirus gene under transcriptional control of a transcriptional response element regulated by said transcriptional transactivator; wherein said transcriptional transactivator is activated by an exogenous inducing agent, the method comprising:

introducing said adenovirus vector into a cell which allows the cell-specific THE to function;
administering said inducing agent;
wherein said adenovirus vector is propagated and causes lysis of said target prostate cell.