EP1866421A2 - Methods and composition related to in vivo imaging of gene expression - Google Patents

Abstract

Disclosed are nucleic acid sequences encoding a recombinant seven transmembrane G-protein associated receptor (GPCR) amino acid sequence operatively coupled to a tissue-selective promoter sequence. Also disclosed are methods of imaging or treating cells in a subject that involve introducing a nucleic acid encoding a reporter detectable in vivo using non-invasive methods operatively coupled to a tissue-selective promoter or amplified tissue specific promoter to the subject, and subjecting the subject to a non-invasive imaging technique or a therapeutic that selectively interacts with the reporter or gene of interest. For example, the subject may be a subject with cancer, and the therapeutic may be an anti-cancer agent. In particular, the hTERT promoter or amplified hTERT promoter drives expression of a reporter such hSSTR2 and/or hSSTR2 derivative) and these may be linked to a gene of interest. Imaging may be performed by a variety of methods including nuclear medicine.

Description

DESCRIPTION

METHODS AND COMPOSITION RELATED TO IN VIVO IMAGING OF GENE

EXPRESSION

BACKGROUND OF THE INVENTION The present application is related to U.S. Provisional Patent Application 60/659,844 filed on March 9, 2005, hereby incorporated by reference in its entirety.

1. Field of the Invention

The present invention relates generally to the fields of imaging, biomedical imaging, molecular biology, gene therapy, and cancer therapy. Aspects of the invention include compositions and methods for non-invasive imaging and/or treatment of a subject. A further aspect of the invention includes a reporter (that may be linked to a gene of interest) that is operatively linked to an amplified tissue specific promoter. A further aspect of the invention includes novel nucleic acid sequences encoding a recombinant seven transmembrane G- protein associated receptor (GPCR) amino acid sequence operatively coupled to a tissue- selective promoter sequence or an amplified tissue specific promoter, including but not limited to hTERT and derivatives thereof.

2. Description of Related Art

Gene therapy and cellular therapies have great promise, but they suffer from a lack of methodology for specifically locating expression of a gene therapy and/or tracking a cell expressing a recombinant nucleic acid within an organism. Various reporter genes can serve to locate and quantify expression of a particular nucleic acid. In a pre-clinical tumor model, the transfer of a reporter gene, a recombinant somatostatin receptor type 2a (SSTR2a) chimera, can be quantified in vivo. A reporter gene may also serve as a weak growth inhibitor when targeting cancer. However, this may not be desirable for other disorders such as diabetes, for cell based therapies, or when evaluating expression and/or localizing of a gene of interest.

Biomedical imaging includes various modalities that are widely used by physicians and researchers to assist with not only the diagnosis of disease in a subject, but also to gain a greater understanding of normal structure and function of the body. One problem with many of these methods is that they either employ radiopharmaceuticals that are not known to be safe for use in humans or they use radioisotopes in ways that may have unforeseen adverse consequences. Another problem is that these methods are limited because transcription of the reporter may be limited due to suboptimal promoter function. Furthermore, imaging may not be targeted to a tissue of interest, such as a tumor.

The combination of more than one imaging modality, such as anatomical and functional imaging technology, would allow for improved resolution and/or characterization of an object or region of interest, such as a tumor.

Therefore, there is the need for reporters that can be delivered to a subject and targeted to a tissue of interest. There is also the need for methods to facilitate reporter imaging technology such as increasing expression. Coupling reporter imaging technology with gene therapy would also allow new methods to monitor efficacy and biodistribution of a second imaging reporter or a therapeutic gene, such as an anticancer gene.

SUMMARY OF THE INVENTION

The ability to non-invasively image expression of a nucleic acid will contribute to the development of gene delivery, such as cancer therapy by providing information on biodistribution of vectors, tissue-related levels of gene expression, expression in the tumor, expression in non-target tissues and the ability to correlate these findings with therapeutic activity. This data will help validate these parameters as predictors of response and provide information that may result in the development of improved vectors and gene expression constructs. Non-invasive molecular imaging requires two components, sensitive/specific reporter molecules and sensitive detection instrumentation. With this technology, new molecular imaging procedures can be developed by integrating new imaging vectors and probes with multimodal-imaging instruments for performing multiple functional-imaging assays.

This technology will facilitate the development of novel molecular imaging approaches specifically designed to assess gene transfer in terms of expression and temporal and spatial distribution as well as therapeutic efficacy. Further, this technology will facilitate the development of dual molecular imaging reporter and therapeutic gene expression vector systems. In certain aspects a tumor-specific promoter-driven therapeutic nucleic acid may be coupled to a reporter, for example, via a bifunctional reporter or via an IRES. These reporter constructs can be combined with various imaging modalities, such as but not limited to gamma camera imaging, to evaluate biodistribution, expression, and/or activity of a gene therapy. In particular aspects regarding cancer, imaging modalities such as, but not limited to MRI and MR spectroscopy can be used to non-invasively monitor tumor growth and response to administration of a therapy and/or gene therapy. In certain embodiments, the gene therapy is administered systemically. These approaches include using imaging probes to identify promoter-driven reporter expression (and thereby simultaneous expression of the therapeutic gene) and other imaging modalities to monitor nucleic acid expression. The use of anatomic imaging such as ultrasound, CT, MRI, MR spectroscopy, can allow for correlation of therapeutic gene expression with tumor regression.

The inventors describe methods of imaging and/or treating cells in a subject. An aspect of the invention includes expression amplification strategies for increasing expression of a reporter and/or a therapeutic in a cell of interest. Examples of such amplification strategies include use of the Gal4VP16 amplification system to amplify expression of a reporter and/or therapeutic, U.S. Patent Application 20030099616, incorporated herein by reference in its entirety. Other amplification strategies utilize tissue selective promoters, such as elements of the hTERT promoter to amplify expression of a reporter in specific cells, tissues, and organs. The reporter may be operatively coupled to one or more genes of interest, such as a therapeutic gene or a recombinant transactivator, and thus can be applied in the evaluation of biodistribution and therapeutic efficacy of the therapeutic gene. Furthermore, the reporter may be operatively coupled to a second reporter.

In certain embodiments, the tissue selective promoter is an hTERT promoter, and the hTERT promoter is operatively coupled to a first reporter. For example, the hTERT promoter sequence may be SEQ ID NO:1. In more particular embodiments, a nucleic acid sequence comprising the hTERT promoter and first reporter may further include a second coding sequence, wherein the second coding sequence encodes a therapeutic, a selectable marker, a recombinant transactivator, or a second imaging gene (reporter). The second coding sequence may be under the control of the hTERT promoter, or under the control of a second promoter. For example, the second promoter may be a CMV promoter operatively coupled to a gene encoding green fluorescent protein (GFP), thus allowing for a nucleic acid that can be imaged by a combination of nuclear medicine-based and light-based imaging modalities.

The reporter may be imaged by single or multiple imaging modalities, such as nuclear based medicine techniques (e.g., PET, SPECT), CT, MRI, MR spectroscopy, ultrasound, and optical imaging modalities. In a further aspect, the reporter may be a fusion protein, such as a receptor protein fused to a tag (including but not limited to hemmaglutinin A). The reporter may be imaged using various techniques including agents that may be or are approved for human use, such as, but not limited to, those labeled with ⁶⁴Cu, ⁶⁸Ga, ¹⁸F, ¹¹¹In or ^99mTc. Various ligands may be labeled with these agents, including but not limited to peptides and analogues thereof (e.g., somatostatin analogues). Certain embodiments of the present invention include a nucleic acid encoding a recombinant seven transmembrane G-protein associated receptor (GPCR) reporter amino acid sequence detectable in a subject by non-invasive methods operatively coupled to a tissue- selective promoter sequence. In some embodiments, the recombinant seven transmembrane G-protein associated receptor has a C-terminus deletion and/or has altered internalization and/or is defective in intracellular signaling. Any amino acid sequence from a GPCR is contemplated for inclusion in the present invention. One of ordinary skill in the art would be familiar with the many GPCRs that have been identified. For example, the GPCR may be an acetylcholine receptor: Ml, M2, M3, M4, or M5; adenosine receptor: Al; A2A; A2B; or A3; adrenoceptors: alphalA, alphalB, alphalD, alpha2A, alpha2B, alpha2C betal, beta2, or beta3; angiotensin receptors: ATI, or AT2; bombesin receptors: BBl, BB2, or BB3; bradykinin receptors: Bl, B2, calcitonin, Ainilin, CGRP, or adrenomedullin receptors; cannabinoid receptors: CBl, or CB2; chemokine receptors: CCRl, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCRlO, CXCRl, CXCR2, CXCR3, CXCR4, CXCR5, CX3CR1, or XCRl; chemotactic receptors : C3a, C5a, or fMLP; cholecystokinin and gastrin receptors: CCKl, or CCK2; corticotropin-releasing factor receptors: CRFl, or CRF2; dopamine receptors: Dl, D2, D3, D4, or D5; endothelin receptors: ET(A) or ET(B); galanin receptors: GALl, GAL2, or GAL3; glutamate receptors: mgll, mgl2, mgl3, mgl4, mgl5, mgl6, mgl7, or mgl8; glycoprotein hormone receptors: FSH, LSH, or TSH; histamine receptors: Hl, H2, H3, or H4; 5-HT receptors: 5-HT1A, 5-HT1B, 5-HT1D, 5-HT1B, 5-HT1F, 5HT2A, 5-HT2F, 5-HT2C, 5-HT3, 5-HT4, 5-HT5A, 5-HT5B, 5-HT6, or 5-HT7; leukotriene receptors: BLT, CysLTl, or CysLT2; lysophospholipid receptors: edgl, edg2, edg3, or edg4; melanocorlin receptors: MCl; MC2; MC3; MC4, or MC5; melatonin receptors: MTl, MT2, or MT3; neuropeptide Y receptors: Yl, Y2, Y4, Y5, or Y6; neurotension receptors: NTSl, or NTS2; opioids: DOP, KOP, MOP, or NOP; P2Y receptors: P2Y1, P2Y2, P2Y4, P2Y6, P2Y11, or P2Y12); peroxisome proliferators: PPAR-alpha, PPAR-beta, or PPAR-gamma; prostanoid receptors: DP, FP, IP, TP, EPl, EP2, EP3, or EP4; protease-activated receptors: PARl, PAR2, PAR3, or PAR4; Somatostatin receptors: SSTRl, SSTR2, SSTR2A, SSTR3, SSTR4, or SSTR5; tachykinin receptors: NKl, NK2, or NK3; thyrotropin-releasing hormone receptors: TRHl, or TRH2; urotensin-II receptor; vasoactivate intestinal peptide or pituitary adenylate cyclase activating peptide receptors: VPACl, VPAC2, or PACl; or vasopressin or oxytocin receptors: Via, VIb, V2, or OT. It is contemplated that other members of the GPCR superfamily may be used in the practice of the invention. In certain particular embodiments, the GPCR is a somatostatin receptor, preferably a somatostatin receptor type 2A (SSTR2A). In some embodiments, the SSTR2A has a C- terminus deletion and/or has altered internalization and/or is signaling defective. In certain particular embodiments, the truncation is carboxy terminal to amino acid 314. A tissue-selective promoter sequence, as used herein, is defined as a promoter that is capable of driving transcription of a nucleic acid in one or more tissues or cell types while remaining largely silent or expressed at relatively low levels in other tissues or cell types. Any tissue-selective promoter sequence known to those of ordinary skill in the art is contemplated by the present invention. A tissue selective promoter sequence may include, but is not limited to a telomerase promoter (human telomerase RNA) (hTR) promoter or human telomerase reverse transcriptase promoter (hTERT) promoter, hi certain aspects of the invention a tissue selective promoter may be used in conjunction with an expression amplification system, as described here. In particular, an amplification system may use an hTERT promoter to drive expression, either directly or indirectly, of a reporter or a reporter and a gene of interest, wherein the gene of interest is operatively coupled to the reporter.

The tissue-selective promoter sequence may be active in one or more tissues or cell types, including but not limited to heart, lung, esophagus, muscle, intestine, breast, prostate, stomach, bladder, liver, spleen, pancreas, kidney, neurons, myocytes, leukocytes, immortalized cells, neoplastic cells, rumor cells, cancer cells, duodenum, jejunum, ileum, cecum, colon, rectum, salivary glands, gall bladder, urinary bladder, trachea, larynx, pharynx, aorta, arteries, capillaries, veins, thymus, mandibular lymph node, mesenteric lymph node, bone marrow, pituitary gland, thyroid gland, parathyroid glands, adrenal glands, brain, cerebrum, cerebellum, medulla, pons, spinal cord, sciatic nerve, skeletal muscle, smooth muscle, bone, testes, epidiymides, prostate, seminal vesicles, penis, ovaries, uterus, mammary glands, vagina, skin, eyes, or optic nerve. In particular aspects a tissue selective promoter will be active in tissue derived from the same embryonic origin or are effected by a similar or the same condition or disease state.

In certain embodiments, the tissue selective promoter is active in a neoplastic cell, a tumor, or a cancer cell. A promoter active in a neoplastic cell, a tumor, or a cancer cell is contemplated for inclusion in the nucleic acids of the present invention. For example, the promoter sequence may be an hTR promoter sequence, hTERT promoter sequence, CEA promoter sequence, a PSA promoter sequence, a probasin promoter sequence, a ARR2PB promoter sequence, an AFP promoter sequence, a MUC-I promoter sequence, a mucin-like glycoprotein promoter sequence, a C-erbB2/neu oncogene promoter sequence, a cyclo- oxygenase promoter sequence, a E2F transcription factor 1 promoter sequence, a tyrosinase related protein promoter sequence, a tyrosinase promoter sequence, or a survivin promoter sequence.

In certain aspects, the promoter sequence is an hTERT promoter sequence that is active in a cancer cell. The cancer cell may be any type of cancer cell, including those derived from mesoderm, endoderm, or ectoderm such as blood, heart, lung, esophagus, muscle, intestine, breast, prostate, stomach, bladder, liver, spleen, pancreas, kidney, neurons, myocytes, leukocytes, immortalized cells, neoplastic cells, tumor cells, cancer cells, duodenum, jejunum, ileum, cecum, colon, rectum, salivary glands, gall bladder, urinary bladder, trachea, larynx, pharynx, aorta, arteries, capillaries, veins, thymus, lymph nodes, , bone marrow, pituitary gland, thyroid gland, parathyroid glands, adrenal glands, brain, cerebrum, cerebellum, medulla, pons, spinal cord, nerves, skeletal muscle, smooth muscle, bone, testes, epidiymides, prostate, seminal vesicles, penis, ovaries, uterus, mammary glands, vagina, skin, eyes, or optic nerve. In further embodiments of the present invention, the promoter sequence is an immunoglobulin heavy chain promoter sequence, an immunoglobulin light chain promoter sequence, a T-cell receptor promoter sequence, an HLA DQ α promoter sequence, an HLA DQ β promoter sequence, a beta-interferon promoter sequence, an interleukin-2 promoter sequence, an interleukin-2 receptor promoter sequence, an MHC Class II 5 promoter sequence, an MHC Class II HLA-Dra promoter sequence, a beta-actin promoter sequence, a muscle creatine kinase (MCK) promoter sequence, a prealbumin (transthyretin) promoter sequence, an elastase I promoter sequence, a metallothionein (MTII) promoter sequence, a collagenase promoter sequence, an albumin promoter sequence, an alpha-fetoprotein promoter sequence, a gamma-globin promoter sequence, a beta-globin promoter sequence, a c-fos promoter sequence, a c-HA-ras promoter sequence, an insulin promoter sequence, a neural cell adhesion molecule (NCAM) promoter sequence, an alpha- 1 -antitrypsin promoter sequence, an H2B (TH2B) histone promoter sequence, a type I collagen promoter sequence, a GRP94 promoter sequence, a GRP78 promoter sequence, an other glucose-regulated protein promoter sequence, a growth hormone promoter sequence, a human serum amyoid A (SAA) promoter sequence, a troponin I (TN I) promoter sequence, a platelet-derived growth factor (PDGF) promoter sequence, a Duchenne Muscular Dystrophy promoter sequence, an SV40 promoter sequence, a polyoma promoter sequence, a retrovirus promoter sequence, a papilloma virus promoter sequence, a Hepatitis B virus promoter sequence, a Human Immunodeficiency Virus promoter sequence, a Cytomegalovirus promoter sequence, a Gibbon Ape Leukemia Virus promoter sequence, a human LIMK2 gene promoter sequence, a somatostatin receptor promoter sequence, a murine epididymal retinoic acid-binding gene promoter sequence, a human CD4 promoter sequence, a mouse alpha2 (XI) collagen promoter sequence, a DlA dopamine receptor promoter sequence, an insulin-like growth factor II promoter sequence, human platelet endothelial cell adhesion molecule- 1 promoter sequence, a human alpha-lactalbumin promoter sequence, a 7SL promoter sequence, a human Y promoter sequence, a human MRP-7-2 promoter sequence, or a 5 S ribosomal promoter sequence or a functional hybrid, functional portion, or a combination of any of tissue specific promoter sequences. Promoters and nucleic acids of the invention may be included in an amplification vector. An amplification vector includes a nucleic acid sequence encoding a transactivator under the control of a tissue specific promoter. The encoded transactivator is coupled or present in a cell with a second nucleic acid including a nucleic acid(s) of interest, such as a reporter or therapeutic, which is under the control of a promoter activated by the transactivator. In certain embodiments the transactivator is a GaIVP 16 transactivator. The GaIVP 16 may include a varying number of GAL and/or VP 16 within the construct. The GaIVP 16 may include one or more genes linked to the GaIVP 16.

The nucleic acids of the present invention may or may not be operatively coupled to a core promoter sequence. A core promoter sequence is defined herein to refer to a nucleotide sequence that maintains the ability to bind and locate a transactivator or a component of a transcription complex to a particular location in a nucleic acid. In some embodiments, tissue- selective promoter sequence is operatively coupled to a core promoter sequence. The core promoter sequence can be any core promoter sequence known to those of ordinary skill in the art. For example, the core promoter sequence may be derived from a ubiquitin promoter, an actin promoter, an elongation factor 1 alpha promoter, an early growth factor response 1 promoter, an eukaryotic initiation factor 4Al promoter, a ferritin heavy chain promoter, a ferritin light chain promoter, a glyceraldehyde 3 -phosphate dehydrogenase promoter, a glucose-regulated protein 78 promoter, a glucose-regulated protein 94 promoter, a heat shock protein 70 promoter, a heat shock protein 90 promoter, a beta-kinesin promoter, a phosphoglycerate kinase promoter, an ubiquitin B promoter, a beta-actin promoter or a minimal viral promoter sequence.

A minimal viral promoter sequence can be any minimal viral promoter sequence known to those of ordinary skill in the art. For example, the minimal viral promoter sequence may be an RNA virus promoter, DNA virus promoter, adenoviral promoter sequence, a baculoviral promoter sequence, a CMV promoter sequence, a parvovirus promoter sequence, a herpesvirus promoter sequence, a poxvirus promoter sequence, an adeno-associated virus promoter sequence, a semiliki forest virus promoter sequence, an SV40 promoter sequence, a vaccinia virus promoter sequence, a lentivirus promoter, a reovirus promoter, or a retrovirus promoter sequence. In particular embodiments, the minimal viral promoter sequence is a mini-CMV promoter sequence. For example, the mini-CMV promoter sequence can be SEQ ID NO:2. In certain aspects of the present invention, the tissue selective promoter is the hTERT promoter. For example, the promoter sequence may include SEQ ID NO:3, which includes an hTERT promoter sequence operatively coupled to a mini-CMV sequence. Various coding sequences are contemplated as the second coding sequence including, but not limited to a therapeutic, a selectable marker, or a second imaging gene (reporter). A therapeutic is defined herein to refer to an agent that is known or suspected to be of benefit in the treatment and/or prevention of a disease in a subject. In a further aspect of the invention a subject can be an animal, preferably a human. A disease can be any disease that can affect a subject, including but not limited to cancer. In particular embodiments, a therapeutic is a tumor suppressor, an inducer apoptosis, an enzyme, an antibody, an antibody fragment, a siRNA, a hormone, a prodrug, or an immunostimulant. hi certain embodiments, the therapeutic is a radiotherapeutic. Any tumor suppressor known to those of ordinary skill in the art is contemplated by the present invention, including but not limited to FUSl, p53, CDK4 or other cyclin-dependent kinase; pl6^B, p21WAFl, CIPI, SDIl, p27^KIP1 or other CDK-inhibitory protein; C-CAM, RB, APC, DCC, NF-I, NF-2, WT-I, MEN-I, MEN- II, zacl, p73, BRCAl, VHL, FCC, MMACl, MCC, plό, p21, p57, p27, and BRCA2. In particular embodiments, the tumor suppressor is FUSl.

A selectable marker is defined herein to refer to a nucleic acid sequence that when expressed confers an identifiable characteristic to the cell permitting easy identification, isolation and/or selection of cells containing the selectable marker from cells without the selectable marker. Any selectable marker known to those of ordinary skill in the art is contemplated for inclusion in the present invention. For example, the selectable marker may be a drug selection marker, an enzyme, an immunologic marker, or a fluorescent protein. In further embodiments, the nucleic acids of the present invention may include a second coding sequence, wherein the second coding sequence and the nucleic acid encoding the recombinant seven transmembrane G-protein associated receptor amino acid sequence are operatively coupled to a bidirectional promoter. Alternatively, the second coding sequence may be under the control of a second promoter. A bidirectional promoter is defined herein to refer to promoter with ability to initiate transcription in both directions from the promoter element. In some embodiments, the second coding sequence and the nucleic acid encoding the recombinant seven transmembrane G-protein associated receptor amino acid sequence are separated by an IRES. An IRES is defined herein to refer to an internal ribosome entry site. The second coding sequence, in certain embodiments, encodes a therapeutic, a selectable marker, or a reporter. As discussed above, the therapeutic can be any therapeutic known to those of ordinary skill in the art. For example, the therapeutic may be a tumor suppressor, an inducer apoptosis, an enzyme, an antibody, an antibody fragment, a siRNA, a hormone, or an immunostimulant. Any tumor suppressor is contemplated for inclusion in the present invention. Exemplary tumor suppressors have been set forth above. Any inducer of apoptosis is contemplated for inclusion in the present invention. Exemplary inducers of apoptosis include TRAIL, Bax, Bak, BcI-X₃, Bik, Bid, Harakiri, Ad ElB, Bad, and ICE-CED2 protease. In certain particular embodiments, the tumor suppressor is FUSl.

As discussed above, any selectable marker known to those of ordinary skill in the art is contemplated for inclusion in the nucleic acid sequences of the present invention. For example, the selectable marker may be a drug selection marker, an enzyme, an immunologic marker, or a fluorescent protein. A drug selection marker is defined herein to as a marker that may be used to select for or against cells that do or do not retain an expressed copy of that marker gene. Exemplary drug selection markers include bacterial aminoglycoside 3' phosphotransferase gene (also referred to as the neo gene) which confers resistance to the drug G418 in mammalian cells, the bacterial hygromycin G phosphotransferase (hyg) gene which confers resistance to the antibiotic hygromycin and the bacterial xanthine-guanine phosphoribosyl transferase gene (also referred to as the gpt gene) which confers the ability to grow in the presence of mycophenolic acid. Other selectable markers are not dominant in that their use must be in conjunction with a cell line that lacks the relevant enzyme activity. Examples of non-dominant selectable markers include the thymidine kinase (tk) gene which is used in conjunction with tk cell lines, the CAD gene which is used in conjunction with CAD- deficient cells and the mammalian hypoxanthine-guanine phosphoribosyl transferase (hprt) gene which is used in conjunction with hprt cell lines. A review of the use of selectable markers in mammalian cell lines is provided in Sambrook et al, (2001). An immunologic marker is defined herein to refers to a polypeptide with a corresponding antibody or antibody fragment that may be used to recognize expression of the polypeptide. Fluorescent proteins are well-known to those of ordinary skill in the art. Examples include, but are not limited to green fluorescent protein (GFP), enhanced green fluorescent protein (EGFP), Renilla Reniformis green fluorescent protein, GFPmut2, GFPuv4, enhanced yellow fluorescent protein (EYFP), enhanced cyan fluorescent protein (ECFP), enhanced blue fluorescent protein (EBFP), citrine and red fluorescent protein from discosoma (dsRED).

In some embodiments, the nucleic acid sequences of the present invention include a protein tag fused to the N-terminal end or C-terminal end of a reporter, in particular a GPCR amino acid sequence. The term "tag," "tag sequence" or "protein tag" refers to a chemical moiety, either a nucleotide, oligonucleotide, polynucleotide or an amino acid, peptide or protein or other chemical, that when added to another sequence, provides additional utility or confers useful properties, particularly in the detection or isolation, of that sequence. Thus, for example, a homopolymer nucleic acid sequence or a nucleic acid sequence complementary to a capture oligonucleotide may be added to a primer or probe sequence to facilitate the subsequent isolation of an extension product or hybridized product. In the case of protein tags, histidine residues (e.g., 4 to 8 consecutive histidine residues) may be added to either the amino- or carboxy-terminus of a protein to facilitate detection, selection, or isolation. Alternatively, amino acid sequences, peptides, proteins or fusion partners representing epitopes or binding determinants reactive with specific antibody molecules or other molecules (e.g., flag epitope, c-myc epitope, transmembrane epitope of the influenza A virus hemaglutinin protein, protein A, cellulose binding domain, calmodulin binding protein, maltose binding protein, chitin binding domain, glutathione S-transferase, and the like) may be added to proteins to facilitate protein isolation, localization, and/or identification by procedures such as affinity or immunoaffinity chromatography, immunohistochemistry, or non-invasive detection methods described herein. Chemical tag moieties include such molecules as biotin, which may be added to either nucleic acids or proteins to facilitate isolation or detection by interaction with avidin reagents, and the like. Numerous other tag moieties are known to, and can be envisioned by the trained artisan, and are contemplated to be within the scope of this definition. The protein tag may, in some embodiments, have enzymatic activity.

Further embodiments of the present invention generally pertain to nucleic acid sequences encoding a reporter that is detectable in a subject by non-invasive methods operatively coupled to a promoter sequence that binds a recombinant transactivator. A recombinant transactivator as defined herein refers to an isolated or an engineered polypeptide that includes a domain (transactivation domain) that induces transcription when positioned appropriately in relation to an appropriate promoter sequence. An engineered transactivator may include a DNA binding domain that recognizes a promoter of interest operably coupled to a transactivation domain. In certain particular embodiments, the recombinant transactivator is Gal4VP16. The nucleic acid may or may not comprise a second coding sequence. In certain embodiments, the reporter encoding sequence and the second coding sequence are separated by an IRES. The second coding sequence may encode a therapeutic, a selectable marker, and/or a reporter. A "reporter," "reporter gene" or "reporter sequence" as used herein refers to any genetic sequence or encoded polypeptide sequence that is detectable and distinguishable from other genetic sequences or encoded polypeptides present in cells. Reporters are discussed at length elsewhere in this specification.

In some embodiments, the nucleic acid sequence further includes a second or third coding sequence operatively coupled to a tissue-selective promoter, wherein the second or third coding sequence encodes a recombinant transactivator.

In certain embodiments of the nucleic acid sequences set forth herein the nucleic acid is comprised in a delivery vehicle. A delivery vehicle is defined herein to refer an entity that associates with a nucleic acid and mediates the transfer of the nucleic acid into a cell. Any delivery vehicle is contemplated by the present invention. For example, the delivery vehicle may include but is not limited to a polypeptide, a lipid, a liposome, a plasmid, a viral vector, a phage, a polyamino acid such as polylysine, a prokaryotic cell, or a eukaryotic cell.

Still further embodiments of the present invention generally pertain to methods of imaging or treating cells in a subject that involve introducing a nucleic acid encoding a reporter detectable in vivo using non-invasive methods operatively coupled to a tissue- selective promoter to a subject; and subjecting the subject to a non-invasive imaging technique or a therapeutic that selectively interacts with the reporter. A therapeutic may be a gene therapy, a radiotherapy, chemotherapy, and/or immunotherapy.

In certain aspects, a tissue selective promoter is active in a neoplastic cell, a tumor cell, or a cancer cell. The tissue selective promoter, in certain embodiments, is active in, for example, a breast cancer cell, a lung cancer cell, a prostate cancer cell, an ovarian cancer cell, a brain cancer cell, a liver cancer cell, a cervical cancer cell, a colon cancer cell, a renal cancer cell, a skin cancer cell, a head and neck cancer cell, a bone cancer cell, an esophageal cancer cell, a bladder cancer cell, a uterine cancer cell, a lymphatic cancer cell, a stomach cancer cell, a pancreatic cancer cell, a testicular cancer cell, a lymphoma cell, or a leukemic cell.

As defined herein, "non-invasive imaging" refers to any method of imaging a tissue in a subject that does not require biopsy or other form of tissue removal from the subject in order to image the tissue. Any method of non-invasive imaging is contemplated by the methods of the present invention. Non-invasive imaging is discussed in greater detail in the specification below. In certain embodiments, non-invasive imaging involves detecting expression of the reporter by assaying for an association between the reporter in cells and a detectable moiety. In some embodiments, the association between the cells expressing the reporter and a detectable moiety involves binding of the detectable moiety by the cells, binding of a ligand operably coupled to the detectable moiety by the cells, cellular uptake of the detectable moiety, or cellular uptake of a ligand operably coupled to the detectable moiety. In certain aspects, non-invasive imaging is by nuclear medicine techniques that afford excellent tissue penetration and require as little as nM quantities of a detectable moiety (generally see Christian et al. , 2004; Ell and Gambhir, 2004).

As defined herein, a detectable moiety is any molecule or agent that can emit a signal that is detectable by non-invasive imaging. For example, the detectable moiety may be a protein, a radioisotope, a fluorophore, a visible light emitting fluorophore, infrared light emitting fluorophore, a metal, a ferromagnetic substance, an electromagnetic emitting substance a substance with a specific MR spectroscopic signature, an X-ray absorbing or reflecting substance, or a sound altering substance. In certain embodiments, the detectable moiety is operably coupled to a ligand that specifically binds the reporter. A "ligand" is defined herein to refer to an ion, a peptide, a oligonucleotide, a molecule, or a molecular group that binds to another chemical entity or polypeptide to form a larger complex. Ligands are discussed in greater detail in the specification below. For example, in some embodiments, the ligand is a nucleic acid, such as a DNA molecule or an RNA molecule, a protein, a polypeptide, a peptide, an antibody, an antibody fragment, or a small molecule. The RNA molecule may be, for example, a siRNA.

Any non-invasive method of imaging known to those of ordinary skill in the art is contemplated by the methods of the present invention. Non-invasive methods of imaging are discussed at length in the specification below. Examples of non-invasive methods include MRI, MR spectroscopy, radiography, CT, ultrasound, planar gamma camera imaging, SPECT, PET, other nuclear medicine-based imaging, optical imaging using visible light, optical imaging using luciferase, optical imaging using a fluorophore, other optical imaging, imaging using near infrared light, or imaging using infrared light.

Certain embodiments of the methods of the present invention further include imaging a tissue during a surgical procedure on a subject. The subject can be any subject, such as a mammal, preferably the subject is a human. In further particular embodiments, the subject is a cancer patient. In these embodiments, the nucleic acid may or may not encode a therapeutic for treating a cancer patient. In some embodiments, the cancer patient is undergoing an anticancer therapy. Exemplary anticancer therapies include chemotherapy, radiation therapy, surgical therapy, immunotherapy, and gene therapy.

As used herein the specification, "a" or "an" may mean one or more. As used herein in the claim(s), when used in conjunction with the word "comprising", the words "a" or "an" may mean one or more than one. As used herein "another" may mean at least a second or more.

Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

FIGs. lA-lC. Expression of the SSTR2A gene chimera assessed in vitro. The ELISA, Western blot and receptor binding assays all demonstrate that 309 cells express more of the reporter fusion protein than 301 cells and no expression of the fusion protein is seen in control cells transfected with vector only. FIG. IA. ELISA of cells transfected with vector (vector) or with the gene chimera (clones 301 and 309). Error bars represent SD of triplicate samples. 30,000 cells were plated per well. * vector vs 301 p<.05, ** 301 vs 309 ρ<.05. FIG. IB. Western blot. Twenty micrograms of protein were loaded per lane. FIG. 1C. Receptor binding assay. Cells (3 x 10⁴ per well) were exposed to 0.1 μM ¹¹¹In-OCt or 0.1 μM ^{π 1}Li-OCt plus 1 μM somatostatin. Error bars represent standard deviation of triplicate samples. Vector, cells transfected with vector; 301 and 309, cells transfected with the gene chimera. * vector vs 301 ρ<.05, ** 301 vs 309 p<.05 FIG. 2. Biodistribution after intravenous administration of ¹¹¹In-OCt. Greater biodistribution of the radiopharmaceutical is seen in tumors derived from 309 cells than 301 cells than cells transfected with vector. Error bars represent standard deviation (n = 6 mice). * vector vs 301 p<.05, ** 301 vs 309 p<.05. FIGs. 3A-3G. γ-camera imaging. By both planar and SPECT imaging, tumors expressing the gene chimera are visible, whereas tumors derived from cells transfected with vector are not. Mice bearing subcutaneous tumors (in both shoulders and right thigh) were injected intravenously with ¹¹¹Li-OCt (13 MBq) and imaged 24 hours later by planar (FIG. 3A) and SPECT (FIG. 3B, coronal; FIG. 3C, axial; FIG. 3D, sagittal planes) imaging. Subcutaneous tumors derived from clone 309 (arrow), 301 (white arrowhead), or vector (yellow arrowhead) transfected cells are in the right shoulder, left shoulder, or right thigh respectively. All three tomographic planes are centered on tumor 309. The planar and SPECT images in this figure are of the same representative mouse. FIG. 3E and FIG. 3F, Counts in excised tumors correlate with measurements derived from ROI analysis of planar (FIG. 3E, n=18) or SPECT (FIG. 3F, n=18) imaging. G, The size of the object on the planar image may not correlate with the true anatomic size of the object. Planar image (top) of phantoms (bottom) of the same size containing 500 microliters of serial 1 :1 dilutions of ¹¹¹In- chloride (93-0.3 μCi). FIGs. 4A-C. MR imaging of a nude mouse. MR defines the anatomic size of the object and can demonstrate its internal morphology. FIG. 4 A, A representative T2 FSE image of a subcutaneous tumor (arrow) in the right thigh. The arrowhead identifies the bladder. FIG. 4B, The weight derived from MR imaging correlates with the weight of excised tumors (n=18). FIG. 4C, A representative T2 FSE image of a subcutaneous tumor in the left shoulder. The arrow points to one of the fluid-debris levels within the tumor. For the MR, the mice were scanned while on their abdomens and the images (FIG. 4A and FIG. 4C) reflect this positioning in order to demonstrate the fluid-debris levels (FIG. 4C). T2 FSE imaging in a 4.7T magnet included the following parameters: TE 4120 msec, TR 72 msec, 4 Nex, field of view 3.5 cm, slice thickness 1 mm with 0.3 mm skip, matrix 256 x 256, resolution 136 microns.

FIGs. 5A-5B. Correlation of uptake by invasive and non-invasive methods. % I.D./g of excised tumors correlates with % I.D./g derived from planar (FIG. 5A, n=18) or SPECT (FIG. 5B, n=18) imaging in combination with MR imaging.

FIGs. 6A-6D. Biodistribution assessed by invasive methods correspond with biodistribution assessed by non-invasive methods. Biodistribution of ¹¹¹In-OCt in excised tumors normalized to tumor weight (FIG. 6A) or MR derived corrected weight (FIG. 6B). Biodistribution of ¹¹¹In-OCt in tumors as assessed by planar (FIG. 6C) or SPECT (FIG. 6D) imaging in combination with MR derived corrected weight. Error bars represent standard deviation (n = 6). Vector, tumors derived from cells transfected with vector; 309 and 301, tumors derived from cells expressing the gene chimera. * vector vs 301 p<.05, ** 301 vs 309 p<.05.

FIGs. 7A-7D. Expression of the SSTR2A gene chimera assessed ex vivo. Ex vivo analysis of representative tumors demonstrates that tumors derived from 309 cells express more of the reporter fusion protein than tumors derived from 301 cells and no expression is seen in tumors derived from control cells transfected with vector only. FIG. 7A. Western blot of tumor tissue using an antibody to the HA tag portion of the fusion protein. 50 micrograms of protein extracted from tumors were loaded per lane.

FIG. 7B, FIG. 7C, FIG. 7D. Immunostaining of tumors using an antibody to the HA tag portion of the fusion protein. (FIG. 7B) Vector, tumors derived from cells transfected with vector; (FIG. 7C) 301 and (FIG. 7D) 309, tumors derived from cells expressing the gene chimera.

FIGs. 8A-8B. Expression of EGFP in various tissues of mice by systemic injection of

EGFP-nanoparticles. FIG. 8A. Lung N417 tumors were established by intrathoracic injection of 10⁶ N417 cells in nude mice. Mice were treated with various EGFP-nanoparticles (20 μg

DNA:40 nmmol DOTAP: Choi/mouse) by i.v. injection. Animals were killed 48 hr after injection and fresh frozen samples of tumor, kidney, spleen, lung, and liver were prepared immediately. Frozen sections were fixed in 4% paraformaldehyde and expression of EGFP was examined immediately under a fluorescence microscope. hTMC, human TERT-mini- CMV promoter. FIG. 8B. Quantification of EGFP expression in tumor and normal mouse tissues by FACS analysis.

FIGs. 9A-9D. FIG. 9 A. Immuno-fiuorescence image analysis of SSRT2A fusion protein expression in HTl 080 transfectant. FIG. 9B. Gamma-camera image of nude mouse bearing s.c. tumors inoculated from corresponding SSRT2A-expressing (A,B,C) and non- expressing (D) cells shown in A. Animals were i.v. injected with 111-In-octreotide (13 MBq) and imaged 24 hr later. FIG. 9C. Coexpression of SSRT2A and FUSl in pLJ290/FUSl- IRES-SSRT2A plasmid transfected H 1299 cells. FIG. 9D. Correlation of radiotracer uptake by tumors exemplified in FIG. 9B. % ID/g = % injected dose per gram.

FIG. 10A-10D. Construction of FUSl and SSRT2A-expressing plasmid vectors for cancer gene therapy and molecular imaging. hTMC, human TERT-mini-CMV promoter. IRES, Internal Ribosome Entry Site. HA, hemmaglutinin domain. HA-SSTR2A with deletion beyond amino acid 314 (delta 314) may replace HA-SSTR2A.

FIG. 11. Map of adenovirus construct for amplified hTERT mediated expression of HA-SSTR2. The construct includes the hTERT promoter, GAL/VP16, a GAL binding site, and a gene chimera consisting of the hemmaglutinin A tag and somatostatin receptor type 2A (HA-SSTR2A) for amplified hTERT mediated expression of HA-SSTR2. HA-SSTR2A with deletion beyond amino acid 314 (delta 314) may replace HA-SSTR2A.

FIG. 12. Telomerase assay demonstrates activity in HTl 080 cells, but not IMR90 cells. The telomerase assay was performed with extracts from HTl 080 cells or IMR90 cells. With HTl 080 cells, the 6 base pair repeat ladder indicates telomerase activity. As a negative control, HTl 080 cell extract was treated with RNase.

FIGs. 13A-13B. Cell membrane subcellular localization is noted of the expressed

HA-S STR2 fusion protein after infection with an adenovirus including a human telomerase promoter and GAL4VP16 amplification system. Relatively increased expression is seen in individual human fibrosarcoma cells, HTl 080 (FIG. 13A), which express telomerase than in human fibroblasts, IMR-90 (FIG. 13B), which do not express telomerase.

FIG. 14. Infection with an adenovirus containing a human telomerase promoter and GALVP16 amplification system results in greater expression of HA-SSTR2A/infected cell in cells that express telomerase (HTl 080, human fibrosarcoma) than in cells that do not express telomerase (IMR-90, human fibroblasts).

FIG. 15. Plasmid map. Driven by an amplified hTMC promoter, expression of both SSTR2A and FUSl are linked. This construct includes the human telomerase promoter (hTERT), a mini cytomegalovirus (miniCMV) promoter, FUSl gene, an internal ribosome entry site, and a gene chimera consisting of hemmaglutinin A tag and a somatostatin receptor type 2 A (HA-S STR2 A) for amplified hTERT mediated expression of FUSl (a gene of interest) linked to HA-SSTR2A.

FIG. 16. The human miniCMV-hTERT (hTMC) promoter increases tissue specific expression by the hTERT promoter. Mice bearing intrathoracic tumor derived from N417 cells were injected intravenously with plasmid-lipoplexes (20 micrograms DNA: 40 nmol

DOTAP:Cholesterol/mouse). Plasmids containing a CMV promoter-enhanced green fluorescent protein (EGFP) resulted in expression in tumor and normal tissues. Plasmids containing an hTERT based promoter resulted in expression in tumors, but not or minimally in normal tissue. The hTMC promoter resulted in greater tumor specific expression than did the unamplified hTERT promoter.

FIG. 17. Driven by a hTMC or a CMV promoter, expression of both SSTR2A and FUSl may be linked. Thus, HA-S STR2 or related genes may be expressed with a gene of interest such as the therapeutic gene FUSl. Lung cancer cells H 1299 transfected with plasmid containing a CMV promoter HA-S STR2 insert results in expression of the HA- SSTR2A fusion protein. H1299 cells transfected with plasmid containing a hTMC promoter FUS1-HA-SSTR2 insert results in expression of both HA-SSTR2A fusion protein and FUSl. H1299 cells transfected with plasmid containing a CMV promoter FUS1-HA-SSTR2 insert results in expression of both HA-SSTR2 fusion protein and FUSl. H1299 cells were transiently transfected and evaluated for fluorescence 72 hours later. The HA-SSTR2 fusion protein was visualized via an antibody targeting the HA-domain and expression is seen at the cell membrane as expected. FUSl was visualized via an antibody to FUSl. The overlay demonstrates colocalization of the nuclear staining and HA-S STR2 (CMV-HA-S STR2) or colocalization of nuclear staining, HA-S STR2, and FUSl when FUSl and HA-S STR2 are linked for expression via an internal ribosome entry site (IRES).

FIGs. 18A-18B. The hTERT promoter amplified by the Gal4-VP16 system results in tumor specific expression in vivo. FIG. 18 A: Nude mouse injected via tail vein with adenovirus containing a CMV promoter-green fluorescent protein insert (negative control, left) does not result in HA-SSTR2A expression in the liver. Normal washout of 111- In-octreotide is seen via the kidneys. Nude mouse injected with adenovirus containing a CMV promoter-HA-SSTR2 insert (positive control, middle) results in expression in the liver. Nude mouse injected with adenovirus containing an hTERT promoter-Gal4/VP16 promoter- HA-S STR2 insert (right) does not result in expression in the liver, thus no expression is seen in normal tissue. FIG. 18B: Nude mouse bearing human fibrosarcoma tumors (derived from HTl 080 cells) was injected intratumorally with adenovirus containing an hTERT promoter- Gal4/VP 16 promoter-HA-SSTR2 insert in the left tumor or with adenovirus containing a CMV promoter-HA-SSTR2 insert in the right tumor. Expression is visualized in both tumors. Mice were injected with virus and two days later injected with 111-In-octreotide. Planar imaging was performed one day later. Representative images are displayed.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

The ability to non-invasively image expression of a nucleic acid will contribute to the development of systemic gene delivery, diagnosing and staging disease, and as a cancer therapy by providing information on biodistribution of vectors, tissue-related levels of gene expression, expression in the tumor, and the ability to correlate these findings with therapeutic activity. This data will help validate these parameters as predictors of response and provide information that may result in the development of improved vectors and gene expression constructs. Non-invasive molecular imaging requires two components, sensitive/specific reporter molecules and sensitive detection instrumentation. With this technology, new molecular imaging procedures can be developed by integrating new imaging vectors and probes with multimodal-imaging instruments for performing multiple functional-imaging assays.

This technology will facilitate the development of novel molecular imaging approaches specifically designed to assess gene transfer in terms of expression and temporal and spatial distribution as well as therapeutic efficacy. Further, this technology will facilitate the development of dual molecular imaging reporter and therapeutic gene expression vector systems. In certain aspects a tumor-specific promoter-driven therapeutic nucleic acid may be coupled to reporter via an IRES. These reporter constructs can be combined with imaging modalities, such as but not limited to gamma camera imaging, to evaluate biodistribution, expression, and/or activity of a gene therapy. In particular aspects regarding cancer, imaging modalities such as, but not limited to MRI and MR spectroscopy can be used to non- invasively monitor tumor growth and response to administration of anticancer therapies, such as gene therapy. In certain embodiments, the gene therapy is administered systemically. These approaches include using imaging probes to identify promoter-driven reporter expression (and thereby simultaneous expression of the therapeutic gene) and other imaging modalities to monitor nucleic acid expression. The use of anatomic imaging such as MRI can allow for correlation of therapeutic gene expression with tumor regression. The use of the tissue specific promoter or amplified telomerase specific promoter and reporter may serve as an agent for diagnosing and staging diseases such as those that are genetic, inflammatory, infectious, or neoplastic. The hTERT promoter (amplified hTERT promoter) and reporter (such as SSTR2 or derivatives) in particular may serve to diagnose and stage cancer.

I. IMAGING OF GENE EXPRESSION

A new form of imaging or molecular imaging that has developed during the past decade involves the in situ or in vivo imaging of a reporter gene. Reporter gene technology was first applied to in situ imaging of tissue sections (reviewed in Blasberg et al, 2003). For example, Hooper et al. (1990) described imaging of luciferase gene expression in single mammalian cells. Certain embodiments of the present invention pertain to methods of imaging cells in a subject using non-invasive imaging techniques. Any imaging modality known to those of ordinary skill in the art is contemplated as a method of detecting expression of the reporter sequence in the present invention.

Reporter imaging has been described as being based on magnetic resonance, nuclear imaging (PET, gamma camera) and/or in vivo optical imaging systems (reviewed in Blasberg et al, 2003). For example, transfer of the herpes simplex virus-1 thymidine kinase or dopamine receptor type-2 has been detected by positron emission tomography (PET) (Alauddin et al, 1996; Alauddin and Conti, 1998; Gambhir et al, 1998; MacLaren et al, 1999; Tjuvajev et al, 1998). In comparison, transfer of the sodium-iodide symporter (Mandell, 1999), dopamine transporter (Auricchio et al, 2003), or the somatostatin receptor type-2 (Kundra, 2002; Sun et al, 2001) has been detected by gamma camera imaging.

In certain embodiments, imaging involves detecting the presence of a reporter, in particular a recombinant GPCR, on the surface of a cell, integrated in the membrane of a cell or in the cytoplasm of a cell. The signal may be detected by using one or more than one imaging modality (discussed in greater detail below). For example, the imaging procedure may be, but is not limited to radiography, ultrasound, CT, MRI, MR spectroscopy, or PET. hi certain aspects, however, the imaging is performed intraoperatively during a surgical procedure on a subject. In certain embodiments, for example, the reporter can be used to selectively label a particular tissue type that the surgeon seeks to excise, hi some embodiments, the tissue to be excised is a tumor tissue, and the intraoperative imaging is imaging of the tumor tissue. Intraoperative imaging may or may not be performed concurrently with administration of a therapeutic gene.

In certain embodiments, imaging is performed concurrently with administration of a therapeutic gene to a subject. For example, a promoter may be operatively coupled to a reporter amino acid sequence and a therapeutic gene. The therapeutic gene can be any type of therapeutic gene known to those of ordinary skill in the art. For example, the therapeutic gene may be a tumor suppressor gene, a gene that induces apoptosis, a gene encoding an enzyme, a gene encoding a structural protein, a gene encoding a receptor, a gene encoding a paracrine factor, a gene encoding antibody, or a gene encoding a hormone. Any therapeutic gene known to those of ordinary skill in the art is contemplated by the methods of the present invention.

Concurrent administration of a reporter amino acid sequence operatively coupled to a therapeutic gene can also be applied in measurement of the biodistribution of a gene in a subject. Thus, the methods of imaging a cell set forth herein can be applied to measure biodistribution of a therapeutic gene in a tissue or in a tumor.

Imaging of the reporter amino acid sequence can be performed by any method known to those of ordinary skill in the art. For example, the reporter may be imaged by administration of a labeled ligand to a subject, wherein the labeled ligand is directed to the reporter amino acid sequence. In other embodiments, the ligand is a radiolabeled probe, such as ^π ^-octreotide. In further embodiments, the ligand is a fluorescent probe that directly or indirectly associates with the reporter amino acid sequence, or an antibody directed against the reporter amino acid sequence. Any method known to those of ordinary skill in the art for measuring a signal derived from a reporter or an associated ligand is contemplated for inclusion in the present invention. Exemplary methods of detecting are as follows.

A. Gamma Camera Imaging

A variety of nuclear medicine techniques for imaging are known to those of ordinary skill in the art. Any of these techniques can be applied in the context of the imaging methods of the present invention to measure a signal from the reporter. For example, gamma camera imaging is contemplated as a method of imaging that can be utilized for measuring a signal derived from the reporter. One of ordinary skill in the art would be familiar with techniques for application of gamma camera imaging. In one embodiment, measuring a signal can involve use of gamma-camera imaging of an ¹¹¹In or ^99mTc conjugate, in particular ¹¹¹In- octreotide or ^99mTc-somatostatin analogue.

B. Computerized Tomography (CT)

Computerized tomography (CT) is contemplated as an imaging modality in the context of the present invention. By taking a series of X-rays, sometimes more than a thousand, from various angles and then combining them with a computer, CT made it possible to build up a three-dimensional image of any part of the body. A computer is programmed to display two-dimensional slices from any angle and at any depth. The slices may be combined to build three-dimensional representations.

In CT, intravenous injection of a radiopaque contrast agent can assist in the identification and delineation of soft tissue masses when initial CT scans are not diagnostic.

Similarly, contrast agents aid in assessing the vascularity of a soft tissue or bone lesion. For example, the use of contrast agents may aid the delineation of the relationship of a tumor and adjacent vascular structures.

CT contrast agents include, for example, iodinated contrast media. Examples of these agents include iothalamate, iohexol, diatrizoate, iopamidol, ethiodol, and iopanoate.

Gadolinium agents have also been reported to be of use as a CT contrast agent (see, e.g. , Henson et al, 2004). For example, gadopentate agents has been used as a CT contrast agent

(discussed in Strunk and Schild, 2004). C. Magnetic Resonance Imaging (MRI)

Magnetic resonance imaging (MRI) is an imaging modality that uses a high-strength magnet and radio-frequency signals to produce images. The most abundant molecular species in biological tissues is water. It is the quantum mechanical "spin" of the water proton nuclei that ultimately gives rise to the signal in imaging experiments and other nuclei can also be imaged. In MRI, the sample to be imaged is placed in a strong static magnetic field (1-12 Tesla) and the spins are excited with a pulse of radio frequency (RF) radiation to produce a net magnetization in the sample. Various magnetic field gradients and other RF pulses then act on the spins to code spatial information into the recorded signals. By collecting and analyzing these signals, it is possible to compute a three-dimensional image which, like a CT image, is normally displayed in two-dimensional slices. The slices may be combined to build three-dimensional representations.

Contrast agents used in MR or MR spectroscopy imaging differ from those used in other imaging techniques. Their purpose is to aid in distinguishing between tissue components with similar signal characteristics and to shorten the relaxation times (which will produce a stronger signal on Tl -weighted spin-echo MR images and a less intense signal on T2-weighted images). Examples of MRI contrast agents include gadolinium chelates, manganese chelates, chromium chelates, and iron particles.

Both CT and MRI provide anatomical information that aid in distinguishing tissue boundaries and vascular structure. Compared to CT, the disadvantages of MRI include lower patient tolerance, contraindications in pacemakers and certain other implanted metallic devices, and artifacts related to multiple causes, not the least of which is motion (Alberico et al, 2004). CT, on the other hand, is fast, well tolerated, and readily available but has lower contrast resolution than MRI and requires iodinated contrast and ionizing radiation (Alberico et al, 2004).

D. PET and SPECT

Imaging modalities that provide information pertaining to information at the cellular level, such as cellular viability, include positron emission tomography (PET) and single- photon emission computed tomography (SPECT). In PET, a patient ingests or is injected with a radioactive substance that emits positrons, which can be monitored as the substance moves through the body.

Closely related to PET is single-photon emission computed tomography, or SPECT. The major difference between the two is that instead of a positron-emitting substance, SPECT uses a radioactive tracer that emits high-energy photons. SPECT is valuable for diagnosing multiple illnesses including coronary artery disease, and already some 2.5 million SPECT heart studies are done in the United States each year.

PET radiopharmaceuticals for imaging are commonly labeled with positron-emitters such as ¹¹C, ¹³N, ¹⁵O, ¹⁸F, ⁸²Rb, ⁶²Cu, and ⁶⁸Ga. SPECT radiopharmaceuticals are commonly labeled with positron emitters such as ^99mTc, ²⁰¹Tl, and ⁶⁷Ga, ¹¹¹In. Regarding brain imaging, PET and SPECT radiopharmaceuticals are classified according to blood-brain-barrier permeability, cerebral perfusion and metabolism, receptor-binding, and antigen-antibody binding (Saha et al, 1994). The blood-brain-barrier (BBB) SPECT agents, such as ^99mTcO4- DTPA, ²⁰¹Tl, and [⁶⁷Ga]citrate are excluded by normal brain cells, but enter into tumor cells because of altered BBB. SPECT perfusion agents such as [¹²³I]IMP, [^99mTc]HMP AO, [^99mTc]ECD are lipophilic agents, and therefore diffuse into the normal brain. Important receptor-binding SPECT radiopharmaceuticals include [¹²³I]QNE, [¹²³I]IBZM, and [¹²³I]iomazenil. These tracers bind to specific receptors, and are of importance in the evaluation of receptor-related diseases

E. Optical Imaging

Optical imaging is another imaging modality that has gained widespread acceptance in particular areas of medicine. Examples include optical labeling of cellular components, and angiography such as fluorescein angiography and indocyanine green angiography. Examples of optical imaging agents include, for example, fluorescein, a fluorescein derivative, indocyanine green, Oregon green, a derivative of Oregon green derivative, rhodamine green, a derivative of rhodamine green, an eosin, an erytlirosin, Texas red, a derivative of Texas red, malachite green, nanogold sulfosuccinimidyl ester, cascade blue, a coumarin derivative, a naphthalene, a pyridyloxazole derivative, cascade yellow dye, dapoxyl dye.

F. Ultrasound

Another biomedical imaging modality that has gained widespread acceptance is ultrasound. Ultrasound imaging has been used noninvasively to provide realtime cross- sectional and even three-dimensional images of soft tissue structures and blood flow information in the body. High-frequency sound waves and a computer create images of blood vessels, tissues, and organs.

Ultrasound imaging of blood flow can be limited by a number of factors such as size and depth of the blood vessel. Ultrasonic contrast agents, a relatively recent development, include perfluorine and perfluorine analogs, which are designed to overcome these limitations by helping to enhance grey-scale images and Doppler signals.

G. Dual Imaging

For example, as set forth above, the imaging modality may include, but are not limited to, CT, MRI, PET, SPECT, ultrasound, or optical imaging. Other examples of imaging modalities known to those of ordinary skill in the art are contemplated by the present invention.

The imaging modalities are performed at any time during or after administration of the composition comprising the diagnostically effective amount of the compound that comprises two imaging moieties. For example, the imaging studies may be performed during administration of the dual imaging compound of the present invention, or at any time thereafter. In some embodiments, the first imaging modality is performed beginning concurrently with the administration of the dual imaging agent, or about 1 sec, 1 hour, 1 day, or any longer period of time following administration of the dual imaging agent, or at any time in between any of these stated times. In some embodiments of the present invention a second imaging modality may be performed concurrently with the first imaging modality, or at any time following the first imaging modality. For example, the second imaging modality may be performed about 1 sec, about 1 hour, about 1 day, or any longer period of time following completion of the first imaging modality, or at any time in between any of these stated times. One of ordinary skill in the art would be familiar with performance of the various imaging modalities contemplated by the present invention.

II. REPORTER SEQUENCES

Both the basic sciences and patient care will benefit from non-invasive methods for following gene expression in vivo. Such tools are needed for vector development and targeting, for assessing genes of interest, and for planning and monitoring therapy. For example, in vivo imaging, using the somatostatin receptor type 2 as a reporter, has demonstrated enhanced infectivity of ovarian cells using an adenovirus containing an alteration of the HI loop compared to wild type virus. Tumor-specific targeting has also been visualized using the carcinoembryonic antigen (CEA) promoter and herpes simplex virus thymidine kinase (HSV-TK) as the reporter. Another application is the in vivo visualization of the induction of two separate genes using a tetracycline-promoter system. Therefore, in vivo monitoring of gene transfer can be approached using reporter genes either alone or in conjunction with a gene of interest. For the latter, the therapeutic gene may be fused to the reporter or be produced as a separate protein. For example, the gene of interest and reporter may be induced by separate promoters in separate delivery vehicles by co-transfection (co- infection) or by separate promoters in the same delivery vehicle . In addition, the two genes may be linked to the same promoter by, for example, an internal ribosome entry site, or a bidirectional promoter. Using such techniques, expression of the gene of interest and reporter correlate. Thus, one may gauge the location, amount, and duration of expression.

Because cells can be transfected with reporter genes, the reporter may be used to follow cell trafficking. For example, in vitro, specific cells may be transfected with a reporter and then returned to an animal to assess homing. In an experimental autoimmune encephalomyelitis model for multiple sclerosis, Costa et al. (2001) transferred myelin basic protein-specific CD4+ T cells that were transduced to express IL- 12 p40 and luciferase. In vivo, luciferase was used to demonstrate trafficking to the central nervous system. In addition, IL- 12 p40 inhibited inflammation. In another system, using positron emission tomography (PET), Koehne et al. (2003) demonstrated in vivo that Epstein-Barr virus (EBV)- specific T cells expressing herpes simplex virus- 1 thymidine kinase (HSV-TK) selectively traffic to EBV+ rumors expressing the T cells' restricting HLA allele. Furthermore, these T cells retain their capacity to eliminate targeted tumors. Capitalizing on sequential imaging, Dubey et al. (2003) demonstrated antigen specific localization of T cells expressing HSV-TK to tumors induced by murine sarcoma virus/Moloney murine leukemia virus (M-MSV/M- MuLV). Tissue specific promoters may also be used to assess differentiation, for example, a stem cell differentiating or fusing with a liver cell and taking up the characteristics of the differentiated cell such as activation of the surfactant promoter in type II pneumocytes. As these examples illustrate, the applications of reporter based imaging are exciting and can find utility in humans. For rapid translation to the clinic, a reporter such as human SSTR2, that can be imaged using FDA approved agents is advantageous. Such ligands are not currently available for the Herpes simplex virus-thymidine kinase (HSV-TK) or the dopamine-2 receptor (D2R), although these are promising reporters. Another possible limitation of HSV-TK is that there may be an immune response to this viral protein, which is not desirable if it is to be used for imaging. Radiopharmaceuticals for imaging HSV-TK and the D2R are almost exclusively PET agents. For PET based systems, an economical production and distribution system would have to be developed or each facility would have to acquire a cyclotron and develop expertise in making the reagents in house. Agents for imaging SSTR2 are already available. These include gamma camera and PET agents. Clinically, PET is significantly more expensive than gamma-camera based imaging and gamma-cameras are far more available world-wide than the more recently produced PET cameras. An imaging reporter also needs to fit into a vector with or without a gene of interest.

For example, adeno-associated virus delivery systems support inserts of approximately 3 kb. The small size of the SSTR2A gene, at 1110 base pairs, allows it to fit into vectors with limited insert space. Another advantage of the SSTR2 based reporters is their location across the cell membrane. Unlike intracellular reporters that require radiopharmaceuticals to cross the cell membrane, reagents for SSTR2 need not penetrate into the cell. This is beneficial for future development of imaging agents targeting SSTR2. An additional benefit of its trans-cell membrane location is accessibility to reagents such as antibodies for live cell sorting. This is important for quickly sorting transfected/infected cells if the reporter is to be used for cell trafficking studies. Other transmembrane reporters are based on pumps, such as the sodium/iodine symporter, as described herein. Although promising, these may not be ideal because their action may alter cell homeostasis.

The SSTR2 has a favorable signaling profile for cancer gene therapy because it is a growth inhibitor. Its ligand, somatostatin, plays a role in regulating a variety of physiologic functions in the brain, pituitary, pancreas, gastrointestinal tract, adrenals, thyroid, kidney and immune system. It inhibits endocrine and exocrine secretions and intestinal motility, as well as modulates neurotransmission, absorption of nutrients and ions, and vascular contractility. Importantly, it acts primarily to inhibit proliferation of normal and tumor cells. The actions of somatostatin are regulated by plasma membrane receptors. The types of receptor and the cellular environment determine the biologic effect of somatostatin. Over-expression of somatostatin receptors can be imaged in vivo and somatostatin receptor based reporters have been proposed for imaging exogenously introduced gene expression. Using the somatostatin receptor as a reporter in a cancer situation is beneficial because it is a weak growth inhibitor and inhibits secretion. The latter is important for symptomatic relief of patients with neuroendocrine tumors such as carcinoid. However, non-target expression of exogenous somatostatin receptor expression may affect other physiologic systems described above. It may also affect the signaling pathways of linked genes of interest. In addition, inhibition of cell growth would not be desirable in cell based therapies where cell implantation and growth is desired. Inhibition of cell growth and secretion would also not be desirable in some gene therapy situations such as diabetes where cells secreting insulin may be a goal. Instead, somatostatin receptor based reporters such as delta 314 are needed that are deficient in signaling but that can still be imaged in vivo.

The term "reporter," "reporter gene" or "reporter sequence" as used herein refers to any genetic sequence or encoded polypeptide sequence that is detectable and distinguishable from other genetic sequences or encoded polypeptides present in cells. Preferably, the reporter sequence encodes a protein that is readily detectable either by its presence, its association with a detectable moiety or by its activity that results in the generation of a detectable signal. In certain aspects, a detectable moiety may include a radionuclide, a fluorophore, a luminophore, a microparticle, a microsphere, an enzyme, an enzyme substrate, a polypeptide, a polynucleotide, a nanoparticle, and/or a nanosphere, all of which may be coupled to an antibody or a ligand that recognizes and/or interacts with a reporter. In various embodiments, a nucleic acid sequence of the invention comprises a reporter nucleic acid sequence or encodes a product that gives rise to a detectable polypeptide. A reporter is or encodes a reporter molecule which is capable of directly or indirectly generating a detectable signal. Generally, although not necessarily, the reporter gene includes a nucleic acid sequence and/or encodes a detectable polypeptide that are not otherwise produced by the cells. Many reporter genes have been described, and some are commercially available for the study of gene regulation (e.g., Alam and Cook, 1990, the disclosure of which is incorporated herein by reference). Signals that may be detected include, but are not limited to color, fluorescence, luminescence, isotopic or radioisotopic signals, cell surface tags, cell viability, relief of a cell nutritional requirement, cell growth and drug resistance. Reporter sequences include, but are not limited to, DNA sequences encoding β-lactamase, β-galactosidase (LacZ), alkaline phosphatase, thymidine kinase, green fluorescent protein (GFP), chloramphenicol acetyltransferase (CAT), luciferase, membrane bound proteins including, for example, G- protein coupled receptors (GPCRs), somatostatin receptors, CD2, CD4, CD8, the influenza hemagglutinin protein, symporters (such as NIS) and others well known in the art, to which high affinity antibodies or ligands directed thereto exist or can be produced by conventional means, and fusion proteins comprising a membrane bound protein appropriately fused to an antigen tag domain from, among others, hemagglutinin or Myc. Aspects of the invention include creating signaling deficient reporters by creating and comparing receptor mutants. However, expressing receptor mutants alone is not ideal for comparing among them, because each may have a different affinity for a ligand, such as octreotide, resulting in confusion between expression levels and affinity. Thus, one first needs to normalize expression independent of reporter binding or immunogenicity. Although a small number of radiopharmaceutical based systems for imaging gene transfer in vivo have been created most of these systems were not designed to directly compare expression independent of the visualized expression product.

A method independent of ligand binding is needed for comparing protein expression and determining intracellular localization. An amino-terminus tag common to all of the mutants allows such comparisons among receptors. Once promising modifications are identified, the epitope tag may be removed, if needed. The inventors have expressed amino- terminal tagged human SSTR2A. The fusion protein detected in vitro, using an antibody to the hemagglutinin-A (HA) amino-terminal tag, correlates with receptor binding studies and biodistribution using ¹¹¹In labeled octreotide. That is, expression levels of the fusion protein in vitro correlate with radiophramaceutical uptake. An additional advantage of the tag is that it can be exploited in an ELISA format for rapidly screening multiple clones for expression or for sorting cells. Further, the tag allows in vitro and ex vivo analysis of expression using an antibody, which is approximately 100 time less expensive than using an imaging radiopharmaceutical. It can be used for ex-vivo studies such as immunohistochemistry of experimental tumors or biopsies and it allows one to separate endogenous versus exogenous human SSTR2. Because the common epitope tag enables comparison of expression levels among mutants without relying simply on ligand binding or antibodies to receptor domains that may be disturbed in mutant receptors, one can create and compare mutants to optimize the reporter. This ability to analyze and correlate findings in vitro, in vivo and ex vivo will also benefit assessing hTERT promoter constructs and amplified hTERT promoter constructs. The inventors have demonstrated that expression of the HA-S STR2 gene chimera can be quantified using in vivo imaging and this corresponds to ex-vivo expression. The functional and anatomic techniques employed small animal version of clinical instruments in order to ease translation of the quantification methodologies to patients.

In certain embodiments, the reporter amino acid sequence is a GPCR. GPCRs are discussed at length elsewhere in this specification. In further embodiments, expression of a reporter nucleic acid or polypeptide confers a growth advantage and the degree of the growth advantage is controllable by varying the growth conditions of the host cell. In other embodiments, a reporter sequence encodes a fluorescent protein. Examples of fluorescent proteins which may be used in accord with the invention include green fluorescent protein (GFP), enhanced green fluorescent protein (EGFP), Renilla Reniformis green fluorescent protein, GFPmut2, GFPuv4, enhanced yellow fluorescent protein (EYFP), enhanced cyan fluorescent protein (ECFP), enhanced blue fluorescent protein (EBFP), citrine and red fluorescent protein from discosoma (dsRED).

In still further embodiments, a reporter nucleic acid may encode a polypeptide having a tag. In association with this embodiment, the method may further comprise the step of contacting the host cell with a fluorescently labeled antibody specific for the tag, thereby labeling the host cell, which may be detected and/or isolated by FACS or other detection, sorting or isolation methods.

In various embodiments, the desired level of expression of at least one of the reporter sequence is an increase, a decrease, or no change in the level of expression of the reporter sequence as compared to the basal transcription level of the reporter sequence. In a particular embodiment, the desired level of expression of one of the reporter sequences is an increase in the level of expression of the reporter sequence as compared to the basal transcription level of the reporter sequence.

In various embodiments, the reporter sequence encodes unique detectable proteins which can be analyzed independently, simultaneously, or independently and simultaneously. In certain embodiments, at least one of the reporter sequence encodes a fluorescent protein.

In other embodiments, the host cell may be a eukaryotic cell or a prokaryotic cell. Exemplary eukaryotic cells include yeast and mammalian cells. Mammalian cells include human cells and various cells displaying a pathologic phenotype, such as cancer cells.

A. Recombinant G-Protein Coupled Receptors (GPCRs)

One class of reporters includes the membrane protein superfamily of seven transmembrane G-protein associated receptors (GPCRs) and its subfamilies. GPCRs are a class of proteins involved in signal transduction, and are one of the largest receptor superfamilies known. These receptors are biologically important, and malfunction of these receptors has been shown to result in diseases such as Alzheimer disease, Parkinson disease, diabetes, dwarfism, color blindness, retinitis pigmentosa and asthma. GPCRs are also involved in depression, schizophrenia, sleeplessness, hypertension, anxiety, stress, renal failure and in several other cardiovascular, metabolic, neural, oncologic and immune disorders (Horn and Vriend, 1998). They have also been shown to play a role in HIV infection (Feng et al, 1996).

GPCRs have been characterized as having seven putative transmembrane domains that are connected by loops. The N-terminus is always extracellular and C-terminus is intracellular. The signal, such as an endogenous ligand or chemical moiety, is received at the extracellular N-terminus side. This signal is then transduced through the membrane to the cytosolic side where a heterotrimeric protein G-protein is activated which in turn elicits a response (see Horn and Vriend, 1998). GPCRs include a wide range of biologically active receptors, such as hormone receptors and neuronal receptors. Examples include, but are not limited to somatostatin receptors, receptors for adrenergic agents and dopamine.

The G-protein family of coupled receptors includes dopamine receptors, which bind to neuroleptic drugs, used for treating psychotic and neurological disorders. Other examples of members of this family of receptors include receptors for calcitonin, endothelin, cAMP, adenosine, acetylcholine, serotonin, histamine, thrombin, kinin, follicle stimulating hormone, opsins, endothelial differentiation gene-1 receptor, rhodopsins, odorant and cytomegalovirus receptors.

Aspects of the invention include noninvasive imaging and/or therapy associated with the introduction of recombinant GPCRs into a cell of interest. Certain embodiments of the present invention generally pertain to nucleic acids encoding recombinant GPCR amino acid sequences operatively coupled to a tissue-selective promoter sequence. Exemplary GPCRs include the acetylcholine receptor: Ml, M2, M3, M4, or M5; adenosine receptor: Al; A2A; A2B; or A3; adrenoceptors: alphalA, alphalB, alphalD, alpha2A, alpha2B, alpha2C betal, beta2, or beta3; angiotensin receptors: ATI, or AT2; bombesin receptors: BBl, BB2, or BB3; bradykinin receptors: Bl, B2, calcitonin, Ainilin, CGRP, or adrenomedullin receptors; cannabinoid receptors: CBl, or CB2; chemokine receptors: CCRl, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCRlO, CXCRl, CXCR2, CXCR3, CXCR4, CXCR5, CX3CR1, or XCRl; chemotactic receptors : C3a, C5a, or fMLP; cholecystokinin and gastrin receptors: CCKl, or CCK2; corticotropin-releasing factor receptors: CRFl, or CRF2; dopamine receptors: Dl, D2, D3, D4, or D5; endothelin receptors: ET(A) or ET(B); galanin receptors: GALl, GAL2, or GAL3; glutamate receptors: mgll, mgl2, mgl3, mgl4, mgl5, mglό, mgl7, or mgl8; glycoprotein hormone receptors: FSH, LSH, or TSH; histamine receptors: Hl, H2, H3, or H4; 5 -HT receptors: 5-HT1A, 5-HT1B, 5-HT1D, 5-HT1B, 5-HT1F, 5HT2A, 5-HT2F, 5-HT2C, 5-HT3, 5-HT4, 5-HT5A, 5-HT5B, 5-HT6, or 5-HT7; leukotriene receptors: BLT, CysLTl, or CysLT2; lysophospholipid receptors: edgl, edg2, edg3, or edg4; melanocorlin receptors: MCl; MC2; MC3; MC4, or MC5; melatonin receptors: MTl, MT2, or MT3; neuropeptide Y receptors: Yl, Y2, Y4, Y5, or Y6; neurotension receptors: NTSl, or NTS2; opioids: DOP, KOP, MOP, or NOP; P2Y receptors: P2Y1, P2Y2, P2Y4, P2Y6, P2Y11, or P2Y12); peroxisome proliferators: PPAR-alpha, PPAR-beta, or PPAR-gamma; prostanoid receptors: DP, FP, IP, TP, EPl, EP2, EP3, or EP4; protease-activated receptors: PARl, PAR2, PAR3, or PAR4; Somatostatin receptors: SSTRl, SSTR2, SSTR2A, SSTR3, SSTR4, or SSTR5; tachykinin receptors: NKl, NK2, or NK3; thyrotropin-releasing hormone receptors: TRHl, or TRH2; urotensin-II receptor; vasoactivate intestinal peptide or pituitary adenylate cyclase activating peptide receptors: VPACl, VPAC2, or PACl; or vasopressin or oxytocin receptors: Via, VIb, V2, or OT.

In certain embodiments, the GPCR is a somatostatin receptor, such as a somatostatin type 2 receptor. Information pertaining to somatostatin receptors can be found in U.S. Patent Application 2002/0173626-A1, which is herein specifically incorporated by reference in its entirety. The nucleic acid encoding the GPCR amino acid sequence may encode an entire

GPCR sequence, a functional GPCR protein domain, a stably expressed non-functional GPCR, a GPCR polypeptide, or a GPCR polypeptide equivalent, each of which may include one or more transmembrane, extracellular, intracellular, extracelullar loop(s) and/or intracellular loop(s). The nucleic acids may be derived from genomic DNA, i.e., cloned directly from the genome of a particular organism, mRNA from a particular organism, and/or synthesized by use of various methods including but not limited to PCR™.

In some embodiments, the nucleic acid may be complementary DNA (cDNA). cDNA is DNA prepared using messenger RNA (mRNA) as a template. Thus, a cDNA does not contain any interrupted coding sequences and usually contains almost exclusively the coding region(s) for the corresponding protein. In other embodiments, the nucleic acid may be produced synthetically.

It may be advantageous to combine portions of the genomic DNA with cDNA or synthetic sequences to generate specific constructs. For example, where an intron is desired in the ultimate construct, a genomic clone may need to be used. Introns may be derived from other genes in addition to GPCR. The cDNA or a synthesized polynucleotide may provide more convenient restriction sites for the remaining portion of the construct and, therefore, would be used for the rest of the sequence.

The present invention includes nucleic acids encoding any reporter or GPCR polypeptide equivalent. These nucleic acids encoding reporter or GPCR polypeptide equivalents may be naturally-occurring homologous nucleic acid sequences from other organisms. A person of ordinary skill in the art would understand that commonly available experimental techniques can be used to identify or synthesize nucleic acids encoding reporter or GPCR polypeptide equivalents. The present invention also encompasses chemically synthesized mutants of these sequences. Another kind of sequence variant results from codon variation. Because there are several codons for most of the 20 normal amino acids, many different DNAs can encode GPCRs. The codons include: Alanine (Ala): GCA, GCC, GCG, and GCU; Cysteine (Cys): UGC and UGU; Aspartic acid (Asp): GAC and GAU; Glutamic acid (GIu): GAA and GAG; Phenylalanine (Phe): UUC and UUU; Glycine (GIy): GGA, GGC, GGG and GGU; Histidine (His): CAC and CAU; Isoleucine (lie): AUA, AUC and AUU; Lysine (Lys): AAA and AAG; Leucine (Leu): UUA, UUG, CUA, CUC, CUG and CUU; Methionine (Met): AUG; Asparagine (Asn): AAC and AAU; Proline (Pro): CCA, CCC, CCG and CCU; Glutamine (GIn): CAA and CAG; Arginine (Arg): AGA, AGG, CGA, CGC, CGG and CGU; Serine (Ser): AGC, AGU, UCA, UCC, UCG and UCU; Threonine (Thr): ACA, ACC, ACG and ACU; Valine (VaI): GUA, GUC, GUG and GUU; Tryptophan (Trp) UGG; Tyrosine (Tyr): UAC and UAU.

Allowing for the degeneracy of the genetic code, sequences that have between about 50% and about 75%, or between about 76% and about 99% of nucleotides that are identical to the nucleic acids disclosed herein will be preferred. Sequences that are within the scope of "a nucleic acid encoding a reporter or GPCR amino acid sequence" are those that are capable of base-pairing with a polynucleotide segment set forth above under intracellular conditions.

As stated above, the reporter or GPCR encoding sequences may be full length genomic or cDNA copies, or fragments thereof. The present invention also may employ shorter oligonucleotides of the reporters or GPCRs. Sequences of 12 bases long should occur only once in the human genome and, therefore, suffice to specify a unique target sequence or PCR oligo. Both binding affinity and sequence specificity of an oligonucleotide to its complementary target increases with increasing length. It is contemplated that oligonucleotides of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 or longer base pairs will be used, for example, in the preparation of GPCR mutants and in PCR reactions. 1 Somatostatin Receptors

There are six somatostatin receptors (SSTR), types 1, 2A and 2B, 3, 4, and 5. Types 2A and 2B are alternate splice variants that are identical, except that type 2A has a longer intracytoplasmic C-terminus. Human type 2 has the highest affinity for the FDA approved somatostatin analogue, ¹¹¹In labeled octreotide. This radiopharmaceutical, approved for whole body imaging, and 99mTc labeled analogues, approved for lung imaging, are used in clinical practice to detect tumors over-expressing somatostatin receptors, such as neuroendocrine tumors. The normal biodistribution and dosimetry of radiolabeled somatostatin analogues used for imaging clinically has been well studied. The radiopharmaceutical is normally found in the kidneys, bladder, liver, spleen and bowel after intravenous injection. At the tracer doses used for imaging, no side-effects greater than placebo are found and patients are routinely imaged serially. Clinically, increased SSTR2 expression renders even small tumors detectable. PET based agents are also being developed. For this receptor, in vitro studies suggest that the sixth and seventh transmembrane domains are essential for binding octreotide. Transmembrane domains three through five may also be important because a cysteine-cysteine disulfide bond is predicted between transmembrane domains three and extracellular domain two. Transmembrane domains three through seven have been predicted to cooperate in forming the pocket for binding octreotide. SSTR2 regulates cAMP production. Gambhir et al. (1999) found that a D2 receptor mutant deficient in regulating cAMP can still be imaged. In COS-7 cells, activation of human SSTR2 results in decreased cAMP production and activation of phospholipase C and calcium mobilization fully or partially, respectively, via a pertussis toxin sensitive G-protein. Through cAMP, somatostatin can regulate secretion. In 32D hematopoietic cells, cAMP appears to be required for SSTR2 mediated chemotaxis. The cytoplasmic C-terminus of the somatostatin receptor is involved in regulating cAMP. Deletion of amino acids beyond 349 of rat SSTR2 increases basal cAMP inhibition in human embryonic kidney (HEK 293) cells. Deletion of amino acids beyond 318 of human SSTR5 eliminates inhibition of cAMP in Chinese hamster ovary (CHO Kl) cells.

Inhibition of proliferation by SSTR2 involves multiple downstream mediators including phosphatases. The tyrosine phosphatase SHP-I is regulated by SSTR2, but SHP-I does not appear to regulate cAMP in the breast carcinoma line MCF-7. Upstream of SHP-I are reported to be inhibitory G proteins, the tyrosine phosphatase SHP-2 and the tyrosine kinase Src. SHP-2 interacts with SSTR2 tyrosine 228 in the context LCYLFI in the third intracellular domain and tyrosine 312 in the context of ILYAFL in transmembrane domain 7 next to the C-terminus. How Src associates with the SSTR2 has not yet been clarified. The phosphatases may have direct effect on phosphorylation of the somatostatin receptor itself, stimulatory growth factors or other downstream effectors. Phosphatidyl inositol, Ras, Rapl, B-raf, MEKl and 2, Map kinase/Erk 1 and 2 have been implicated in SSTR2 mediated signaling in CHO DG44 cells; but in neuroblastoma cells, Ras did not appear to be involved and Map kinase/Erk 1 and 2 activity decreased, instead of increased as in CHO DG44 cells. Thus, the role of the MAP kinase pathway in mediating inhibition of proliferation by SSTR2 is not yet clear. Also downstream of SHP-I is the neuronal nitric oxide synthase (nNOS) and guanylate cyclase, both of which appeal" necessary for SSTR2 mediated inhibition of proliferation in CHO cells and mouse pancreatic acinar cells. The inhibition may also involve other phosphotyrosine phosphatases such as r-PTPη and more downstream effectors such as cyclin dependent kinase inhibitor p27kipl. Somatostatin also regulates transcription factors such as c-jun, c-fos and AP-I .

For signaling, the C-terminus and intracytoplasmic domains of SSTR2 appear to be involved. As stated above, for both rat SSTR2 and human SSTR5, deletion analysis has demonstrated that the cytoplasmic C-terminus regulates inhibition of cAMP production. In particular, deletion of the SSTR2 after amino acid 314 is signaling defective and can be imaged in vivo.

B. Detecting Reporters of the Invention In certain embodiments of the invention, a reporter may be imaged by detecting its association with a ligand. A ligand is defined herein to refer to an ion, a peptide, a oligonucleotide, aptamer, a molecule, or a molecular group that binds to another chemical entity or polypeptide to form a larger complex. In the context of the present invention, the ligand may bind to a reporter or to an amino acid sequence attached to the reporter sequence (e.g., such as a protein tag fused to the N-terminal end or C-terminal end of the reporter amino acid sequence) to form a larger complex. Any ligand known to those of ordinary skill in the art is contemplated for use as a ligand in the context of the present invention. In some embodiments of the present invention, a ligand may be contacted with the cell for imaging. The ligand may or may not be internalized by the cell. Where a reporter has become localized to the cell surface, the ligand, in these embodiments, may bind to or associate with the reporter. Any method of binding of the ligand to the reporter is contemplated by the present invention. In certain other embodiments, a ligand may become internalized by a cell. Once internalized the ligand may, but need not, bind to or associate with the reporter or a second reporter within the cell. The ligand may be a molecule or part of a molecule that has properties or is conjugated to a moiety such that it is capable of generating a signal that can be detected. Any imaging modality known to those of ordinary skill in the art can be applied to image a ligand. In some embodiments, the ligand is capable of binding to or being coupled to a molecule or part of a molecule that can be imaged. For example, the ligand may be capable of binding to or be coupled to a radionuclide, and the radionuclide can be imaged using nuclear medicine techniques known to those of ordinary skill in the art. For example, the ligand may be ¹¹¹In- octreotide. In other embodiments, for example, the ligand is capable of binding to or being coupled to a contrast agent that can be detected using imaging techniques well-known to those of ordinary skill in the art. For example, the ligand may be capable of binding to or being coupled to a CT contrast agent or an MRI contrast agent.

In certain embodiments of the present invention, a ligand can bind to the reporter, and the ligand in turn generates a signal that can be measured using an imaging modality known to those of ordinary skill in the art. In other embodiments, the ligand can bind to a protein tag that is fused to the reporter. Thus, for example, imaging would involve measuring a signal from the ligand, and this in turn would provide for localization of the reporter sequence within the cell or within a subject.

A variety of valent metal ions, or radionuclides, are known to be useful for radioimaging. Examples include, but are not limited to ⁶⁷Ga, ⁶⁸Ga, "'"Tc, ¹¹¹In, ¹²³I, ¹²⁵1, ¹³¹I, ¹⁶⁹Yb, ⁶⁰Cu, ⁶¹Cu, ⁶⁴Cu, ⁶²Cu, ²⁰¹Tl, ⁷²A, and ¹⁵⁷Gd. Due to better imaging characteristics and lower price, attempts have been made to replace the ¹²³I, ¹³¹I, ⁶⁷Ga and ¹¹¹In labeled compounds with corresponding ^99mTc labeled compounds when possible. Due to favorable physical characteristics as well as low price, ^PPmTc has been preferred to label radiopharmaceuticals .

A number of factors must be considered for optimal radioimaging in humans. To maximize the efficiency of detection, a valent metal ion that emits gamma energy in the 100 to 200 keV range is preferred. A "gamma emitter" is herein defined as an agent that emits gamma energy of any range. One of ordinary skill in the art would be familiar with the various valent metal ions that are gamma emitters. To minimize the absorbed radiation dose to the patient, the physical half-life of the radionuclide should be as short as the imaging procedure will allow. To allow for examinations to be performed on any day and at any time of the day, it is advantageous to have a source of the radionuclide always available at the clinical site. ^mTc is a preferred radionuclide because it emits gamma radiation at 140 keV, it has a physical half-life of 6 hours, and it is readily available on-site using a molybdenum- 99/technetium-PPm generator. One of ordinary skill in the art would be familiar with methods to determine optimal radioimaging in humans.

In some embodiments of the compositions of the present invention, a valent metal ion that is a therapeutic metal ion that is not a beta emitter or a gamma emitter can be bound to the ligand or reporter amino acid sequence. For example, the therapeutic metal ion may be platinum, cobalt, copper, arsenic, selenium and thallium. Compounds including these therapeutic metal ions may be applied in the methods of the present invention directed to the treatment of hyperproliferative disease, such as the treatment of cancer. The imaging agents described herein may be used to localize a radio therapy to a cell expressing a nucleic acid of the invention.

In certain embodiments of the present invention, the nucleic acid for use in the imaging methods of the present invention encodes an amino acid sequence that can be radiolabeled in vivo. Radiolabeling of the encoded reporter sequence can be direct, or it can be indirect, such as by radiolabeling of a ligand that can bind the protein tag or reporter sequence. Radiolabeled agents, compounds, and compositions provided by the present invention are provided having a suitable amount of radioactivity. For example, in forming ^99mTc radioactive complexes, it is generally preferred to form radioactive complexes in solutions containing radioactivity at concentrations of from about 0.01 millicurie (mCi) to about 300 mCi per mL.

Once the encoded sequence is radiolabled, it can be imaged for visualizing a site, such as a tumor in a mammalian body. In accordance with this invention, the radiolabel is administered by any method known to those of ordinary skill in the art. For example, administration may be in a single unit injectable dose, administered as a radiolabeled ligand. Any of the common carriers known to those with skill in the art, such as sterile saline solution or plasma, may be utilized. Generally, a unit dose to be administered has a radioactivity of about 0.01 mCi to about 300 mCi, preferably 5 mCi to about 30 mCi. The solution to be injected at unit dosage is usually from about 0.01 mL to about 10 mL. After intravenous administration of the radiolabeled reagent, imaging of the organ or tumor in vivo can take place, if desired, in minutes, hours or even longer, after the radiolabeled reagent is introduced into a patient. In some instances, a sufficient amount of the administered dose will accumulate in the area to be imaged within about 0.1 of an hour.

As set forth above, imaging may be performed using any method known to those of ordinary skill in the art. Examples include PET, SPECT, and gamma scintigraphy. In gamma scintigraphy, the radiolabel is a gamma-radiation emitting radionuclide and the radiotracer is located using a gamma-radiation detecting camera (this process is often referred to as gamma scintigraphy). The imaged site is detectable because the radiotracer is chosen either to localize at a pathological site (termed positive contrast) or, alternatively, the radiotracer is chosen specifically not to localize at such pathological sites (termed negative contrast).

C. Therapies Diagnosis, Staging, and Imaging Targets

There is great promise for gene therapy and cellular therapies to treat a variety of diseases such as cardiovascular diseases, genetic diseases, diabetes, neurodegenerative diseases, and cancer. Methods for monitoring and specifically imaging expressed genes and cell trafficking are in their infancy. Most new gene and cell therapies will target specific tissues. However, it remains difficult to locate gene expression in the body without performing a biopsy. Clearly, tagging cells or a gene with a reporter that can be non- invasively imaged would be beneficial for monitoring efficacy and toxicity. Expression of the reporter can be linked to expression of a gene of interest. One would then be able to determine whether expression is achieved in the target tissue and, with the appropriate reporter, determine whether the necessary level of expression or functional cell engraftment is achieved for therapeutic effect. Additionally, if the reporter could be repeatedly visualized, the duration and level of expression in a particular location could be repeatedly monitored, aiding dosing regimens. Maintaining long-term expression is presently a challenge for the field of gene therapy. Following expression in non-target tissues should also prove fruitful, because of its potential to impact toxicity. For cancer therapy, a reporter that is also an inhibitor of growth is desirable, but for other disease such as diabetes, a reporter that causes as little perturbation to the cell as possible is more desirable. Even for cancer, a reporter that is signaling deficient may be more desirable in order to limit effects on non-target tissues and to limit cross-talk with a linked gene of interest.

Currently most imaging techniques utilize anatomic change in order to diagnose and stage disease. Functional imaging methods that depend on a biologic process can identify disease before there is an anatomic change or can be used to understand the etiology of an anatomic change resulting in a specific diagnosis. For example, the bone scan agent 99m-Tc- MDP can identify bone metastases before radiography or computed tomography (Rieden, 1995) or if a bone lesion is found on anatomic imaging, increased uptake of 99m-Tc-MDP or 18-fluorine-deoxyglucose (18-FDG) PET can help separate malignancy from a benign lesion. 18-FDG PET imaging has proven useful for diagnosing and staging some, but not all malignancies. For example, renal cell carcinomas (Kang et al, 2004) and prostate cancer (Salminen et al, 2002; Hofer et al, 1999), are not well differentiated by 18-FDG-PET and inflammatory lesions can mimic cancer (Albes et al, 2002). 18-FDG-PET mimics glucose, thus, reflects metabolism. Another approach to identify disease is to use disease or tissue specific promoters. Telomerase activity is not found or is present in low levels in normal tissues, but is present in almost all cancers (Kim et al, 1994; Broccoli et al, 1995), for example, those arising in the reproductive organs (ICyo et al, 1996), breast (Hiyama et al, 1996), colon (Chadeneau et al, 1995), liver (Tahara et al, 1995), brain (Langford et al, 1995), prostate (Sommerfeld et al, 1996), and lung ( Hiyama et al, 1995). Human telomerase (hTERT) promoter activity localizes with telomerase activity (Takakura et al, 1999; Cong et al, 1999). Thus, the hTERT promoter may be used to specifically localize gene expression in tumors. Typically, tissue specific promoters are too weak for therapy, so amplification methods may be needed. An amplified hTERT promoter could be used to drive expression of a reporter in cancer for diagnosis and staging as well as to drive expression of a reporter linked to a therapeutic gene of interest.

Thus, imaging methods designed to detect gene expression in vivo may be needed for further development of gene delivery systems, for diagnosing disease, as well as for monitoring clinical efficacy and toxicity. Further, a number of imaging systems will be needed for monitoring either single gene therapy or multiple gene therapies in a patient. For example, a patient with diabetes and cancer may benefit from two different gene therapy interventions that can be monitored independently by two separate reporters. If an inducible promoter is used with a reporter, it may be advantageous to also transfer a constitutively active promoter driving a different reporter to gauge whether gene transfer occurred in case induction fails. Although a few gene expression imaging systems have been designed, many do not sufficiently penetrate human tissues (those based on light such as GFP or luciferase) for percutaneous imaging and most do not utilize radiopharmaceuticals, which are proven safe in humans and are FDA approved.

The present invention contemplates methods of preventing, inhibiting, or treating such diseases or conditions in a subject by administration of a nucleic acid encoding a reporter detectable in vivo using non-invasive methods operatively coupled to a tissue-selective promoter, and subjecting the subject to a therapeutic or imaging agent that selectively interacts with the reporter. Aspects of the invention also include the use of the methods and compositions of the invention in combination with other anticancer therapies. Diseases to be prevented, treated or diagnosed can be any disease that affect a subject that would be amenable to therapy or prevention through administration of a therapeutic as described herein. The disease may be a hyperproliferative disease. A hyperproliferative disease is a disease associated with the abnormal growth or multiplication of cells. The hyperproliferative disease may be a disease that manifests as lesions in a subject. Exemplary hyperproliferative lesions include pre-malignant lesions, cancer, and tumors. The cancer can be any type of cancer including those derived from mesoderm, endoderm, or ectoderm such as blood, heart, lung, esophagus, muscle, intestine, breast, prostate, stomach, bladder, liver, spleen, pancreas, kidney, neurons, myocytes, leukocytes, immortalized cells, neoplastic cells, tumor cells, cancer cells, duodenum, jejunum, ileum, cecum, colon, rectum, salivary glands, gall bladder, urinary bladder, trachea, larynx, pharynx, aorta, arteries, capillaries, veins, thymus, lymph nodes, bone marrow, pituitary gland, thyroid gland, parathyroid glands, adrenal glands, brain, cerebrum, cerebellum, medulla, pons, spinal cord, nerves, skeletal muscle, smooth muscle, bone, testes, epidiymides, prostate, seminal vesicles, penis, ovaries, uterus, mammary glands, vagina, skin, eyes, or optic nerve.

Other examples of diseases to be treated include return of lost or lack of function such as diabetes where insulin production is inadequate, infectious diseases, genetic diseases, and inflammatory diseases, such as autoimmune diseases. The methods and compositions of the present invention can be applied to deliver an antigen that can be applied in immune therapy or immune prophylaxis of a disease. One of ordinary skill in the art would be familiar with the many disease entities that would be amenable to prevention or treatment using the pharmaceutical compositions and methods set forth herein.

Treating cells in a subject can involve "inhibiting the growth" of a hyperproliferative lesion, such as cancer, in a subject. "Inhibiting the growth" is broadly defined and includes, for example, a slowing or halting of the growth of the lesion. Inhibiting the growth of a lesion can also include a reduction in the size of a lesion or induction of apoptosis of the cells of the lesion. Induction of apoptosis refers to a situation wherein a drug, toxin, compound, composition or biological entity bestows apoptosis, or programmed cell death, onto a cell. In a specific embodiment, the cell is a tumor cell. Growth of a lesion can also be inhibited by induction of an immune response against the cells of the lesion.

The therapeutic can be any agent that can be applied in the prevention or treatment of any disease in a subject. For example, the therapeutic can be an anti-cancer agent. In some embodiments, the therapeutic is a radionuclide. Radionuclides are discussed in greater detail below. In other embodiments, the therapeutic is an immunomodulator. The imaging agent may be used to localize a therapeutic to the cells or area surrounding the cells expressing the reporter of the invention.

III. NUCLEIC ACIDS

Aspects of the invention include the delivery of a nucleic acid encoding a polypeptide that may be non-invasively imaged by direct or indirect detection. Nucleic acids of the invention include, but are not limited to recombinant GPCRs that produce or bind to or enzymatically act upon agents that produce a detectable signal. In other aspects, nucleic acids may include one or more additional nucleic acid sequences, which include but are not limited to nucleic acid sequence encoding a transactivator, encoding a therapeutic or encoding a second imaging agent.

The term "nucleic acid" is well known in the art. A "nucleic acid" as used herein will generally refer to a molecule (i.e., a strand) of DNA, RNA or a derivative or analog thereof, comprising a nucleobase. A nucleobase includes, for example, a naturally occurring or derivatized purine or pyrimidine base found in DNA (e.g., an adenine "A," a guanine "G," a thymine "T" or a cytosine "C") or RNA (e.g., an A, a G, an uracil "U" or a C). The term "nucleic acid" encompass the terms "oligonucleotide" and "polynucleotide," each as a subgenus of the term "nucleic acid." The term "oligonucleotide" refers to a molecule of between about 3 and about 100 nucleobases in length. The term "polynucleotide" refers to at least one molecule of greater than about 100 nucleobases in length.

These definitions generally refer to a single-stranded molecule, but in specific embodiments will also encompass an additional strand that is partially, substantially or fully complementary to the single-stranded molecule. Thus, a nucleic acid may encompass a double-stranded molecule or a triple-stranded molecule that comprises one or more complementary strand(s) or "complement(s)" of a particular sequence comprising a molecule

The term "vector" is used to refer to a carrier into which a nucleic acid sequence can be inserted for introduction into a cell where it can be expressed and/or replicated. The term "expression vector" or "nucleic acid vector" refers to a nucleic acid containing a nucleic acid sequence or "cassette" coding for at least part of a nucleic acid sequence, also referred to herein as a gene, product capable of being transcribed and "regulatory" or "control" sequences, which refer to nucleic acid sequences necessary for the transcription and possibly translation of an operably linked coding sequence in a particular host cell, hi addition to control sequences that govern transcription and translation, expression vectors may contain nucleic acid sequences that serve other functions as well.

A. Expression Vectors

1 Promoters

The term "promoter" is used interchangeably with "promoter element" and "promoter sequence." Likewise, the term "enhancer" is used interchangeably with "enhancer element" and "enhancer sequence." A promoter, enhancer, or repressor, is said to be "operably linked" to a nucleic acid or transgene, such as a nucleic acid encoding a recombinant seven transmembrane G-protein associated receptor, when such element(s) control(s) or affect(s) nucleic acid or transgene transcription rate or efficiency. For example, a promoter sequence located proximally to the 5' end of a transgene coding sequence is usually operably linked with the transgene. As used herein, term "regulatory elements" is used interchangeably with "regulatory sequences" and refers to promoters, enhancers, polyadenylation sites and other expression control elements, or any combination of such elements. Promoters are positioned 5' (upstream) to the genes that they control. Many eukaryotic promoters contain two types of recognition sequences: TATA box and the upstream promoter elements. The TATA box, located 25-30 bp upstream of the transcription initiation site, is thought to be involved in directing RNA polymerase II to begin RNA synthesis at the correct site. In contrast, the upstream promoter elements determine the rate at which transcription is initiated. These elements can act regardless of their orientation, but they must be located within 100 to 200 bp upstream of the TATA box.

Enhancer elements can stimulate transcription up to 1000-fold from linked homologous or heterologous promoters. Enhancer elements often remain active even if their orientation is reversed (Li et al, 1990). Furthermore, unlike promoter elements, enhancers can be active when placed downstream from the transcription initiation site, e.g., within an intron, or even at a considerable distance from the promoter (Yutzey et al, 1989).

As is known in the art, some variation in this distance can be accommodated without loss of promoter function. Similarly, the positioning of regulatory elements with respect to the transgene may vary significantly without loss of function. Multiple copies of regulatory elements can act in concert. Typically, an expression vector comprises one or more enhancer sequences followed by, in the 5' to 3' direction, a promoter sequence, all operably linked to a transgene followed by a polyadenylation sequence.

A "promoter" sequence is a control sequence that is a region of a nucleic acid sequence at which initiation and rate of transcription are controlled. It may contain genetic elements at which regulatory proteins and molecules may bind such as RNA polymerase and other transcription factors. The phrases "operatively positioned," "operatively linked," "under control," and "under transcriptional control" mean that a promoter is in a correct functional location and orientation in relation to a nucleic acid sequence to control transcriptional initiation and expression of that sequence. A promoter may or may not be used in conjunction with an "enhancer," which refers to a cis-acting regulatory sequence involved in the transcriptional activation of a nucleic acid sequence. Together, an appropriate promoter or promoter/enhancer combination, and a gene of interest, comprise an expression cassette. One or more expression cassettes may be present in a given nucleic acid vector or expression vector. In certain aspects, one expression cassette may encode a transactivator that interacts with a promoter of a second expression cassette. The one or more expression cassettes may be present on the same and/or different expression vector.

A promoter may be one naturally associated with a gene or sequence, as may be obtained by isolating a portion the 5' non-coding sequences located upstream of the coding segment or exon. Such a promoter can be referred to as "endogenous." Similarly, an enhancer may be one naturally associated with a nucleic acid sequence, located either downstream or upstream of that sequence. Alternatively, certain advantages will be gained by positioning the nucleic acid segment under the control of a recombinant or heterologous promoter, which refers to a promoter that is not normally associated with a nucleic acid sequence in its natural environment. In certain . aspect of the invention a heterologous promoter may be a chimeric promoter, where elements of two or more endogenous, heterologous or synthetic promoter sequences are operatively coupled to produce a recombinant promoter.

A recombinant or heterologous enhancer refers also to an enhancer not normally associated with a nucleic acid sequence in its natural environment. Such promoters or enhancers may include promoters or enhancers of other genes, and promoters or enhancers isolated from any other prokaryotic, viral, or eukaryotic cell, and promoters or enhancers not "naturally occurring," i.e., containing different elements of different transcriptional regulatory regions, and/or mutations that alter expression. In addition to producing nucleic acid sequences of promoters and enhancers synthetically, sequences may be produced using recombinant cloning and/or nucleic acid amplification technology, including PCR™, in connection with the compositions disclosed herein (see U.S. Patents 4,683,202 and 5,928,906, each incorporated herein by reference). Such promoters may be used to drive reporter expression, which include, but are not limited to GPCRs, β-galactosidase or luciferase to name a few. Furthermore, it is contemplated the control sequences that direct transcription and/or expression of sequences within non-nuclear organelles such as mitochondria, chloroplasts, and the like, can be employed as well.

A promoter and/or enhancer will typically be used that effectively directs the expression of the DNA segment in a cell type, organelle, and organism chosen for expression. Those of skill in the art of molecular biology generally know the use of promoters, enhancers, and cell type combinations for protein expression, for example, see Sambrook et al, (2001), incorporated herein by reference. The promoters employed may be constitutive, tissue- specific, inducible, and/or useful under the appropriate conditions to direct expression of the introduced DNA segment, such as is advantageous in the production of recombinant proteins and/or peptides. The promoter may be heterologous or endogenous or a combination thereof.

2 Tissue-Selective Promoter Sequences

Most gene transfer methods use promoters such as the cytomegalovirus promoter in order to obtain high levels of expression in a variety of tissues. If introducing a toxic gene, for example to treat a cancer, it would be desirable to specifically target the tumor and not normal tissues. Reporter technology can be used to demonstrate target specificity of tissue specific promoters in vivo.

A disadvantage of tissue specific promoters, including hTERT, is that their ability to drive transcription is relatively weak. Amplification schemes are needed. An amplified promoter active in a variety of cancers driving a reporter can serve to localize and quantify expression of a linked therapeutic gene. An amplified promoter and reporter only combination may be used to gauge therapy induced alterations in hTERT promoter function. This combination also has the potential to serve as a diagnostic tool for identifying and staging cancer. To limit effects on normal cells due to promoter leakiness, a signaling deficient reporter is desirable in such a construct.

In a preferred embodiment the tissue specific promoter is the telomerase promoter. Unlike cancer cells, normal somatic cells have a restricted life span. With each cell division, the ends of chromosomes, called telomeres, become smaller. This is because the RNA primer that is necessary to prime DNA replication is degraded at the 5' end. Therefore, no replication occurs at the telomeres and the chromosome shortens with each replication cycle. Eventually, critical genes are affected and the cell stops growing or dies. Because tumor cells divide indefinitely, they must overcome the end replication problem. This is most often accomplished by activating the ribozyme telomerase, which adds nucleotides, in units of six base pair repeats, to the ends of DNA, lengthening the telomere, thus, increasing the cell's life-span.

Telomerase activity is not found or is present in low levels in normal tissues, but is present in approximately 85-90% of all cancers, for example, those arising in the reproductive organs, breast, colon, liver, brain, prostate, and lung. In vitro and in animal models, many therapies designed to inhibit the telomerase ribozyme have shown some promise. A limitation of these approaches may be that it may take multiple doubling times for telomeres' length to shorten enough to cause cell senescence or death. Others have used portions of the telomerase ribozyme as antigens to create tumor specific T cells. Although there are some limitations, particularly for brain tumors, this is also a promising approach. There are two major components involved in telomerase activity. The first is the template for the 6-base pair repeat sequence, human telomerase RNA (hTR), which is expressed in almost every cell. The second is the rate-limiting step for telomerase activity, human telomerase reverse transcriptase (hTERT), whose promoter activity localizes with telomerase activity.

Thus, the hTERT promoter has been exploited as a means to specifically deliver therapeutic genes to cancer and for restricting replication of oncolytic viruses to malignancies. Maintaining adequate levels of gene expression for therapeutic effect is a challenge for gene/viral therapy and has proven difficult with most tissue or cancer specific promoters. It has also been possible by PET to image a low level of expression of the sodium iodide symporter driven by the hTERT promoter after intratumoral injection of adenovirus. In tumors derived from A2780 cells (ovarian carcinoma), expression was low, including lower than from the ubiquitously expressing hTR promoter or the ubiquitously expressing CMV promoter. No significant expression from the hTERT was seen in tumors derived from PancO2 cells (pancreatic carcinoma), although expression was driven by the hTR and the CMV promoters. Thus, hTERT promoter activity was weak or non-existent in this imaging experiment.

Amplification strategies are needed. Expression from tissue/cancer specific promoters can be enhanced. The GAL4/VP16 system has been used to drive therapeutic gene expression from the carcinoembryonic antigen (CEA) promoter and more recently, the hTERT promoter. Adenovirus incorporating the latter combined with apoptosis inducing genes TRAIL or Bax resulted in tumor killing, but avoided liver or systemic toxicity associated with adenoviruses incorporating TRAIL or Bax driven by a CMV promoter. Thus, it is possible to enhance expression by the hTERT promoter and still maintain cancer specific expression. Enhanced expression from weak promoters can also be attempted by other means, such as the Cre/loxP system, which requires excision of a stuffer DNA and is dependent upon the transcriptional activity of a viral promoter that may be cell dependent. Another method is to use a tissue specific promoter to drive a gene of interest and an artificial transcriptional activator, which then stimulates the promoter. This results in expression of the gene of interest and transcription factor. An additional strategy is to place the hTERT promoter upstream of a ubiquitous promoter such as CMV as exemplified in this application. Amplified hTERT promoters would not only be useful for driving expression of therapeutic genes, but may also be useful for following oncolytic virus restricted to replication in tumors by hTERT based promoters. Linking an imagable reporter such as, but not limited to those based on the somatostatin receptor type 2, would allow localization and quantification of expression needed to obtain a therapeutic effect and to analyze if inappropriate expression is causing toxicity. Because hTERT is almost universally expressed in tumors, when amplified and linked to the reporter, the vector system also has the potential to detect, aid differential diagnosis, and stage tumors. Thus, an imaging system incorporating an amplified hTERT promoter driving a reporter alone, or driving a reporter linked to a gene of interest should prove useful for diagnosis, staging, and therapy. Amplification schemes can also be applied to boost expression of other tissue specific promoters such as those targeting beta cells for treating diabetes or others targeting tumors, such as the CEA promoter for treating malignancy. As stated above, the somatostatin receptor is a weak growth inhibitor and, therefore, may augment cancer therapy. Importantly, every cell in a tumor does not need to express the gene for imaging; if some cells express the reporter, it should be enough for diagnosis and staging, or to use the reporter to follow expression of a linked gene.

The position of the promoter/gene, promoter/reporter, and/or promoter/transactivator may be varied. It is contemplated that 1, 2, 3, 4, or more expression cassettes may be present in a particular vector or a particular cell with no general preference as to the order of the cassettes in an expression vector. A first, second, third or fourth promoter of an expression cassette may be tissue specific, amplified tissue specific, and/or constitutive promoter that drives expression of a gene of interest, reporter, and/or a transactivator. The order of placement of the promoter and gene of interest before or after the promoter complex and reporter is not important for the invention.

Low levels of telomerase activity have also been detected in normal cells, including stem cells. Hematopoietic stem cells tend to be resistant to infection by a variety of vectors. Most stem cells are quiescent and as such are resistant to infection/transfection by most vectors, so that at any one time only a few stem cells would have the potential for infection. Lymphocytes can transiently have increased telomerase activity when undergoing clonal expansion, but again only this small population would have the potential for infection and high level expression via an exogenously introduced hTERT promoter. Quiescent, primitive stem cells have low telomerase activity and the hTERT promoter is much less active in CD34+ hematopoietic progenitor cells. Another method to limit toxicity if there is leaky expression from the hTERT promoter is to use a signaling deficient reporter. Thus, untoward events in cells will be limited and there will be less potential for cross-talk with a gene of interest if the a signaling deficient reporter is employed with an amplified hTERT promoter. Certain aspects of the invention include promoter sequences that interact with endogenous or exogenous transactivators. In certain aspects a transactivator is a recombinant transactivator. A recombinant transactivator may be expressed in cells into which a nucleic acid of the invention is introduced. Alternatively, a recombinant transactivator or a nucleic acid encoding a recombinant transactivator may be introduced before, with or after a nucleic acid of the invention. In certain aspects, the recombinant transactivator may be encoded in a nucleic acid encoding an imaging or therapeutic agent.

A promoter may be functional in a variety of tissue types and in several different species of organisms, or its function may be restricted to a particular species and/or a particular normal or diseased tissue or cell type. Further, a promoter may be constitutively active, or it may be selectively activated by certain substances {e.g., a tissue-specific factor), under certain conditions {e.g., hypoxia, or the presence of an enhancer element in the expression cassette containing the promoter), or during certain developmental stages of the organism {e.g., active in fetus, silent in adult). Promoters useful in the practice of the present invention are preferably tissue-specific-

-that is, they are capable of driving transcription of a gene in one or a few normal or diseased tissue(s) while remaining largely "silent" or expressed at relatively low levels in other tissue types. It will be understood, however, that tissue-specific or tissue-selective promoters may have a detectable amount of "background" or "base" activity in those tissues where they are silent. The degree to which a promoter is selectively activated in a target tissue can be expressed as a selectivity ratio (activity in a target tissue/activity in a control tissue). In this regard, a tissue specific promoter useful in the practice of the present invention typically has a selectivity ratio of greater than about 1 :1.01, 1 :1.1, 1 :1.5 , 1 :2, 1 :3, 1 :4, 1 :5 or more. Preferably, the selectivity ratio is greater than about 1:1.5. The promoter may also function in a reverse manner with decreased activity in the normal or diseased tissue(s) of interest.

It will be further understood that certain promoters, while not restricted in activity to a single tissue type, may nevertheless show selectivity in that they may be active in one group of tissues, and less active or silent in another group. Such promoters are also termed "tissue specific" or "tissue selective," and are contemplated for use with the present invention. For example, promoters that are active in a variety of tumor cells may be therapeutically useful in treating cancer, which may effect any of a number of different regions of the body.

Tissue-specific promoters may be derived, for example, from promoter regions of genes that are differentially expressed in different normal or diseased tissues or at different stages of growth or in cells that are hyperplastic. The level of expression of a gene under the control of a particular promoter can be modulated by manipulating the promoter region. For example, different domains within a promoter region may possess different gene-regulatory activities. The roles of these different regions are typically assessed using vector constructs having different variants of the promoter with specific regions deleted (i.e., deletion analysis) or base pair(s) mutated. Vectors used for such experiments typically contain a reporter sequence, which is used to determine the activity of each promoter variant under different conditions. Application of such a deletion analysis enables the identification of promoter sequences containing desirable activities and thus identifying a particular promoter domain, including core promoter elements, those elements when deleted detrimentally effect characteristics of the promoter, such as but not limited to selectivity or transcription factor binding. This approach may be used to identify, for example, the smallest region capable of conferring tissue specificity, or the smallest region conferring a robust transcriptional response when combined with other promoter elements, such as but not limited to the core CMV promoter or a mini-CMV. A number of tissue specific promoters, described herein, may be particularly advantageous in practicing the present invention, hi most instances, these promoters may be isolated as convenient restriction digest fragments suitable for cloning into a selected vector. Certain aspects the invention include, but are not limited to the hTERT promoter. Alternatively, promoter fragments may be isolated using the polymerase chain reaction or by oligonucleotide synthesis. Cloning of these promoter fragments may be facilitated by incorporating restriction sites at the 5' ends of the primers.

One of ordinary skill in the art would be familiar with the various types of tissue- selective promoter sequences that can be included in the context of the present invention. An exemplary list of these promoters/enhancers includes, but is not limited to Immunoglobulin Heavy Chain (Banerji et al, 1983; Grilles et al, 1983; Grosschedl et al, 1985; Atchison et al, 1986, 1987; Imler et al, 1987; Weinberger et al, 1984; Kiledjian et al, 1988; Porton et al; 1990); Immunoglobulin Light Chain (Queen et al, 1983; Picard et al, 1984); T-CeIl Receptor (Luria et al, 1987; Winoto et al, 1989; Redondo et al; 1990); HLA DQ α and/or DQ β (Sullivan et al, 1987); β-Interferon (Goodbourn et al, 1986; Fujita et al, 1987; Goodbourn et al, 1988); Interleukin-2 (Greene et al, 1989); Interleukin-2 Receptor (Greene et al, 1989; Lin et al, 1990); MHC Class II 5 (Koch et al, 1989); MHC Class II HLA-Dra (Sherman et al, 1989); β-Actin (Kawamoto et al, 1988; Ng et al; 1989); Muscle Creatine Kinase (MCK) (Jaynes et al, 1988; Horlick et al, 1989; Johnson et al, 1989); Prealbumin (Transthyretin) (Costa et al, 1988; Elastase I, Ornitz et al, 1987); Metallothionein (MTII) (Karin et al, 1987; Culotta et al, 1989); Collagenase (Pinkert et al, 1987; Angel et al, 1987); Albumin (Pinkert et al, 1987; Tranche et al, 1989, 1990); α-Fetoprotein (Godbout et al, 1988; Campere et al, 1989; γ-Globin, Bodine et al, 1987; Perez-Stable et al, 1990); β-Globin (Trudel et al, 1987); c-fos (Cohen et al, 1987); c-HA-røs (Treisman, 1986; Deschamps et al, 1985); Insulin (Edlund et al, 1985); Neural Cell Adhesion Molecule (NCAM) (Hirsch et al, 1990); Ot₁ -Antitrypsin (Latimer et al, 1990); H2B (TH2B) Histone (Hwang et al, 1990); Mouse and/or Type I Collagen (Ripe et al, 1989); Glucose-Regulated Proteins (GRP94 and GRP78) (Chang et al, 1989); Rat Growth Hormone (Larsen et al, 1986); Human Serum Amyloid A (SAA) (Edbrooke et al, 1989); Troponin I (TN I) (Yutzey et al, 1989); Platelet- Derived Growth Factor (PDGF) (Pech et al, 1989); Duchenne Muscular Dystrophy (Klamut et al, 1990); SV40 (Banerji et al, 1981; Moreau et al, 1981; Sleigh et al, 1985; Firak et al, 1986; Herr et al, 1986; Imbra et al, 1986; Kadesch et al, 1986; Wang et al, 1986; Ondek et al, 1987; Kuhl et al, 1987; Schaffher et al, 1988); Polyoma (Swartzendruber et al, 1975; Vasseur et al, 1980; Katinka et al, 1980, 1981; Tyndall et al, 1981; Dandolo et al, 1983; de Villiers et al, 1984; Hen et al, 1986; Satake et al, 1988; Campbell and Villarreal, 1988); Retroviruses (Kriegler et al, 1982, 1983; Levinson et al, 1982; Kriegler et al, 1983, 1984a, b, 1988; Bosze et al, 1986; Miksicek et al, 1986; Celander et al, 1987, 1988; Thiesen et al, 1988; Choi et al, 1988; Reisman et al, 1989); Papilloma Virus (Campo et al, 1983; Lusky et al, 1983; Spandidos and Wilkie, 1983; Spalholz et al, 1985; Lusky et al, 1986; Cripe et al, 1987; Gloss et al, 1987; Hirochika et al, 1987; Stephens et al, 1987); Hepatitis B Virus (Bulla et al, 1986; Jameel et al, 1986; Shaul et al, 1987; Spandau et al, 1988; Vannice et al, 1988); Human Immunodeficiency Virus (Muesing et al, 1987; Hauber et al, 1988; Jakobovits et al, 1988; Feng et al, 1988; Takebe et al, 1988; Rosen et al, 1988; Berkhout et al, 1989; Laspia et α/., 1989; Sharp et al, 1989; Braddock et al, 1989); Cytomegalovirus (CMV) (Weber et al, 1984; Boshart et al, 1985; Foecking et al, 1986); and/or Gibbon Ape Leukemia Virus (Holbrook et al, 1987; Quinn et al, 1989).

3 Internal Ribosome Entry Sites (IRES)

In certain embodiments of the invention, the use of internal ribosome entry sites (IRES) elements are used to create multigene, or polycistronic, messages. IRES elements are able to bypass the ribosome scanning model of 5' methylated Cap dependent translation and begin translation at internal sites (Pelletier and Sonenberg, 1988). IRES elements from two members of the picornavirus family (polio and encephalomyocarditis) have been described (Pelletier and Sonenberg, 1988), as well an IRES from a mammalian message (Macejak and Sarnow, 1991) and further sequences as well as modified versions are envisioned in this application for invention. IRES elements can be linked to heterologous open reading frames. Multiple open reading frames can be transcribed together, each separated by an IRES, creating polycistronic messages. By virtue of the IRES element, each open reading frame is accessible to ribosomes for efficient translation. Multiple genes can be efficiently expressed using a single promoter/enhancer to transcribe a single message (U.S. Patents 5,925,565 and 5,935,819; and PCT application PCT/US99/05781) and are envisioned in this application for invention. The order (upstream or downstream of the IRES) of the reporter and gene(s) of interest is not important for the invention. More than one gene of interest may be linked.

4 Selectable Markers

In certain embodiments of the invention, a nucleic acid construct of the present invention may be isolated or selected for in vitro or in vivo by including a selectable marker in the expression vector. Such selectable markers would confer an identifiable characteristic to the cell permitting easy identification, isolation and/or selection of cells containing the expression vector. A positive selectable marker is one in which the presence of the marker allows for its selection, while a negative selectable marker is one in which its presence prevents its selection. An example of a positive selectable marker is a drug resistance marker. Examples of selectable and screenable markers are well known to one of skill in the art. 5 Other Elements of Expression Cassettes

Most transcribed eukaryotic RNA molecules will undergo RNA splicing to remove introns from the primary transcripts. Vectors containing genomic eukaryotic sequences may require donor and/or acceptor splicing sites to ensure proper processing of the transcript for protein expression (Chandler et al, 1997). One may include a polyadenylation signal in the expression construct to effect proper polyadenylation of the transcript. The nature of the polyadenylation signal is not believed to be crucial to the successful practice of the invention, and/or any such sequence may be employed. Specific embodiments include the SV40 polyadenylation signal and/or the bovine growth hormone polyadenylation signal, convenient and/or known to function well in various target cells. Also contemplated as an element of the expression cassette is a transcriptional termination site. These elements can serve to enhance message levels and/or to minimize read through from the cassette into other sequences.

The vectors or constructs of the present invention may comprise at least one termination signal. A "termination signal" or "terminator" is comprised of the DNA sequences involved in specific termination of an RNA transcript by an RNA polymerase. Thus, in certain embodiments a termination signal that ends the production of an RNA transcript is contemplated. A terminator may be necessary in vivo to achieve desirable message levels. In eukaryotic systems, the terminator region may also comprise specific DNA sequences that permit site-specific cleavage of the new transcript so as to expose a polyadenylation site. This signals a specialized endogenous polymerase to add a stretch of about 200 A residues (polyA) to the 3' end of the transcript. RNA molecules modified with this polyA tail appear to more stable and are translated more efficiently. Thus, in other embodiments involving eukaryotes, it is preferred that the terminator comprises a signal for the cleavage of the RNA, and it is more preferred that the terminator signal promotes polyadenylation of the message. The terminator and/or polyadenylation site elements can serve to enhance message levels and to minimize read through from the cassette into other sequences. Terminators contemplated for use in the invention include any known terminator of transcription described herein or known to one of ordinary skill in the art, including but not limited to, for example, the termination sequences of genes, such as for example the bovine growth hormone terminator or viral termination sequences, such as for example the SV40 terminator. In certain embodiments, the termination signal may be a lack of transcribable or translatable sequence, such as due to a sequence truncation.

In order to propagate a vector in a host cell, it may contain one or more origins of replication sites (often termed "ori"), which is a specific nucleic acid sequence at which replication is initiated. Alternatively an autonomously replicating sequence (ARS) can be employed if the host cell is yeast.

B. Nucleic Acid and Genes

Certain embodiments of the present invention pertain to nucleic acid sequences that include one or more nucleic acid sequences of interest, also referred to as genes of interest. For example, a nucleic acid encoding a recombinant GPCR may be operatively coupled to one or more nucleic acids of interest. Certain other embodiments of the present invention pertain to methods of imaging a cell, wherein the methods are further defined as methods of delivering a nucleic acid encoding an imaging component to a cell. Any nucleic acid or gene known to those of ordinary skill in the art is contemplated for inclusion in the methods of delivering a nucleic acid to a cell. The term "gene" is used for simplicity to refer to a functional protein, polypeptide, or peptide-encoding unit and does not necessarily refer to a genomic fragment including the exon and introns of genoniically encoded gene. Thus, gene is used to denote a nucleic acid that includes a nucleotide sequence that includes all or part of a nucleic acid sequence associated with a particular genetic locus. Aspects of the invention include nucleic acids or genes that encode a detectable and/or therapeutic polypeptide, hi certain embodiments of the present invention, the gene is a therapeutic, or therapeutic gene. A "therapeutic gene" is a gene which can be administered to a subject for the purpose of treating or preventing a disease. For example, a therapeutic gene can be a gene administered to a subject for treatment or prevention of diabetes or cancer. Examples of therapeutic genes include, but are not limited to, Rb, CFTR, pi 6, p21, p27, p57, p73, C-CAM, APC, CTS-I, zacl, scFV ras, DCC, NF-I, NF-2, WT-I, MEN-I, MEN-II, BRCAl, VHL, MMACl, FCC, MCC, BRCA2, IL-I, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-I l IL- 12, GM-CSF, G-CSF, thymidine kinase, mda7, fusl, interferon α, interferon β, interferon γ, ADP, p53, ABLI, BLCl, BLC6, CBFAl, CBL, CSFIR, ERBA, ERBB, EBRB2, ETSl, ETS2, ETV6, FGR, FOX, FYN, HCR, HRAS, JUN, KRAS, LCK, LYN, MDM2, MLL, MYB, MYC, MYCLl, MYCN₅ NRAS, PIMl, PML, RET, SRC, TALI, TCL3, YES, MADH4, RBl, TP53, WTl, TNF, BDNF, CNTF, NGF, IGF, GMF, aFGF, bFGF, NT3, NT5, ApoAI, ApoAIV, ApoE, Rapl A, cytosine deaminase, Fab, ScFv, BRCA2, zacl, ATM, HIC-I, DPC-4, FHIT, PTEN, INGl, NOEYl, NOEY2, OVCAl, MADR2, 53BP2, IRF-I, Rb, zacl, DBCCR-I, rks-3, COX-I, TFPI, PGS, Dp, E2F, ras, myc, neu, raf, erb. fms, trk, ret, gsp, hst, abl, ElA, p300, VEGF, FGF, thrombospondin, BAI-I, GDAIF, or MCC.

In certain embodiments of the present invention, the therapeutic gene is a tumor suppressor gene. A tumor suppressor gene is a gene that, when present in a cell, reduces the tumorigenicity, malignancy, or hyperproliferative phenotype of the cell. This definition includes both the full length nucleic acid sequence of the tumor suppressor gene, as well as non-full length sequences of any length derived from the full length sequences. It being further understood that the sequence includes the degenerate codons of the native sequence or sequences which may be introduced to provide codon preference in a specific host cell. Examples of tumor suppressor nucleic acids within this definition include, but are not limited to APC, CYLD, HIN-I, KRAS2b, plό, pl9, p21, p27, p27mt, p53, p57, p73, PTEN, Rb, Uteroglobin, Skp2, BRCA-I, BRCA-2, CHK2, CDKN2A, DCC, DPC4, MADR2/JV18, MENl, MEN2, MTSl, NFl, NF2, VHL, WRN, WTl, CFTR, C-CAM, CTS-I, zacl, scFV, MMACl, FCC, MCC, Gene 26 (CACNA2D2), PL6, Beta* (BLU), Luca-1 (HYALl), Luca-2 (HYAL2), 123F2 (RASSFl), 101F6, Gene 21 (NPRL2), or a gene encoding a SEM A3 polypeptide and FUSl. Other exemplary tumor suppressor genes are described in a database of tumor suppressor genes at www.cise.ufl.edu/~yyl/HTML-TSGDB/Homepage.litml. This database is herein specifically incorporated by reference into this and all other sections of the present application. Nucleic acids encoding tumor suppressor genes, as discussed above, include tumor suppressor genes, or nucleic acids derived therefrom {e.g., cDNAs, cRNAs, mRNAs, and subsequences thereof encoding active fragments of the respective tumor suppressor amino acid sequences), as well as vectors comprising these sequences. One of ordinary skill in the art would be familiar with tumor suppressor genes that can be applied in the present invention.

In certain embodiments of the present invention, the therapeutic gene is a gene that induces apoptosis (i.e., a pro-apoptotic gene). A "pro-apoptotic gene amino acid sequence" refers to a polypeptide that, when present in a cell, induces or promotes apoptosis. The present invention contemplates inclusion of any pro-apoptotic gene known to those of ordinary skill in the art. Exemplary pro-apoptotic genes include CD95, caspase-3, Bax, Bag- 1, CRADD, TSSC3, bax, hid, Bak, MKP-7, PERP, bad, bcl-2, MSTl, bbc3, Sax, BIK, BID, and mda7. One of ordinary skill in the art would be familiar with pro-apoptotic genes, and other such genes not specifically set forth herein that can be applied in the methods and compositions of the present invention. The therapeutic gene can also be a gene encoding a cytokine. The term 'cytokine' is a generic term for proteins released by one cell population which act on another cell as intercellular mediators. A "cytokine" refers to a polypeptide that, when present in a cell, maintains some or all of the function of a cytokine. This definition includes full-length as well as non-full length sequences of any length derived from the full length sequences. It being further understood, as discussed above, that the sequence includes the degenerate codons of the native sequence or sequences which may be introduced to provide codon preference in a specific host cell.

Examples of such cytokines are lymphokines, monokines, growth factors and traditional polypeptide hormones. Included among the cytokines are growth hormones such as human growth hormone, N-methionyl human growth hormone, and bovine growth hormone; parathyroid hormone; thyroxine; insulin; proinsulin; relaxin; prorelaxin; glycoprotein hormones such as follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH), and luteinizing hormone (LH); hepatic growth factor; prostaglandin, fibroblast growth factor; prolactin; placental lactogen, OB protein; tumor necrosis factor-α and -β; mullerian-inhibiting substance; mouse gonadotropin-associated peptide; inhibin; activin; vascular endothelial growth factor; integrin; thrombopoietin (TPO); nerve growth factors such as NGF-β; platelet- growth factor; transforming growth factors (TGFs) such as TGF-α and TGF-β; insulin-like growth factor-I and -II; erythropoietin (EPO); osteoinductive factors; interferons such as interferon-α, -β, and -γ; colony stimulating factors (CSFs) such as macrophage-CSF (M-CSF); granulocyte-macrophage-CSF (GM-CSF); and granulocyte-CSF (G-CSF); interleukins (ILs) such as IL-I, IL-lα, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-IO IL-I l, IL-12; IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-24 LIF, G- CSF₅ GM-CSF, M- CSF, EPO, kit-ligand or FLT-3. Other examples of therapeutic genes include genes encoding enzymes. Examples include, but are not limited to, ACP desaturase, an ACP hydroxylase, an ADP-glucose pyrophorylase, an ATPase, an alcohol dehydrogenase, an amylase, an amyloglucosidase, a catalase, a cellulase, a cyclooxygenase, a decarboxylase, a dextrinase, an esterase, a DNA polymerase, an RNA polymerase, a hyaluron synthase, a galactosidase, a glucanase, a glucose oxidase, a GTP ase, a helicase, a hemicellulase, a hyaluronidase, an integrase, an invertase, an isomerase, a kinase, a lactase, a lipase, a lipoxygenase, a lyase, a lysozyme, a pectinesterase, a peroxidase, a phosphatase, a phospholipase, a phosphorylase, a polygalacturonase, a proteinase, a peptidease, a pullanase, a recombinase, a reverse transcriptase, a topoisomerase, a xylanase, a reporter gene, an interleukin, or a cytokine. Further examples of therapeutic genes include the gene encoding carbamoyl synthetase I, ornithine transcarbamylase, arginosuccinate synthetase, arginosuccinate lyase, arginase, fumarylacetoacetate hydrolase, phenylalanine hydroxylase, alpha-1 antitrypsin, glucose-6-phosphatase, low-density-lipoprotein receptor, porphobilinogen deaminase, factor VIII, factor IX, cystathione beta.-synthase, branched chain ketoacid decarboxylase, albumin, isovaleryl-CoA dehydrogenase, propionyl CoA carboxylase, methyl malonyl CoA mutase, glutaryl CoA dehydrogenase, insulin, beta.-glucosidase, pyruvate carboxylase, hepatic phosphorylase, phosphorylase kinase, glycine decarboxylase, H-protein, T-protein, Menkes disease copper-transporting ATPase, Wilson's disease copper-transporting ATPase, cytosine deaminase, hypoxanthine-guanine phosphoribosyltransferase, galactose- 1 -phosphate uridyltransferase, phenylalanine hydroxylase, glucocerbrosidase, sphingomyelinase, α-L- iduronidase, glucose-6-phosphate dehydrogenase, HSV thymidine kinase, or human thymidine kinase. Therapeutic genes also include genes encoding hormones. Examples include, but are not limited to, genes encoding growth hormone, prolactin, placental lactogen, luteinizing hormone, follicle-stimulating hormone, chorionic gonadotropin, thyroid-stimulating hormone, leptin, adrenocorticotropin, angiotensin I, angiotensin II, β-endorphin, β-melanocyte stimulating hormone, cholecystokinin, endothelin I, galanin, gastric inhibitory peptide, glucagon, insulin, lipotropins, neurophysins, somatostatin, calcitonin, calcitonin gene related peptide, β-calcitonin gene related peptide, hypercalcemia of malignancy factor, parathyroid hormone-related protein, parathyroid hormone-related protein, glucagon-like peptide, pancreastatin, pancreatic peptide, peptide YY, PHM, secretin, vasoactive intestinal peptide, oxytocin, vasopressin, vasotocin, enkephalinamide, metorphinamide, alpha melanocyte stimulating hormone, atrial natriuretic factor, amylin, amyloid P component, corticotropin releasing hormone, growth hormone releasing factor, luteinizing hormone-releasing hormone, neuropeptide Y, substance K, substance P, or thyrotropin releasing hormone.

As will be understood by those in the art, the term "therapeutic gene" includes genomic sequences, cDNA sequences, and smaller engineered gene segments that express, or may be adapted to express, proteins, polypeptides, domains, peptides, fusion proteins, and mutants. The nucleic acid molecule encoding a therapeutic gene may comprise a contiguous nucleic acid sequence of about 5 to about 12000 or more nucleotides, nucleosides, or base pairs. "Isolated substantially away from other coding sequences" means that the gene of interest forms part of the coding region of the nucleic acid segment, and that the segment does not contain large portions of naturally-occurring coding nucleic acid, such as large chromosomal fragments or other functional genes or cDNA coding regions. Of course, this refers to the nucleic acid segment as originally isolated, and does not exclude genes or coding regions later added to the segment by human manipulation.

Encompassed within the definition of "therapeutic gene" is a "biologically functional equivalent" therapeutic gene. Accordingly, sequences that have about 70% to about 99% homology of amino acids that are identical or functionally equivalent to the amino acids of the therapeutic gene will be sequences that are biologically functional equivalents provided the biological activity of the protein is maintained.

C. Delivery of a Nucleic Acids

Aspects of the invention include detecting nucleic acids delivered alone or in combination with a second imaging agent and/or a therapeutic agent. In certain aspects the nucleic acid delivered includes a recombinant GPCR of the invention and in particular embodiments a SSTR.

1 Viral Vectors

Certain embodiments of the present invention pertain to compositions that include a delivery vehicle for delivery of the nucleic acid into a cell. One of ordinary skill in the art would understand use of delivery vehicles for delivery of nucleic acids to a cell since these experimental methods are well-known in the art. In particular, techniques using "viral vectors" are well-known in the art. A viral vector is meant to include those constructs containing viral sequences sufficient to (a) support packaging of the expression cassette and (b) to ultimately express a recombinant gene construct that has been cloned therein.

One method for delivery of a nucleic acid involves the use of an adenovirus vector. Adenovirus vectors are known to have a low capacity for integration into genomic DNA. Adenovirus vectors result in highly efficient gene transfer.

Adenoviruses are currently the most commonly used vector for gene transfer in clinical settings. Among the advantages of these viruses is that they are efficient at gene delivery to both nondividing and dividing cells and can be produced in large quantities. The vector comprises a genetically engineered form of adenovirus (Grunhaus et ah, 1992). In contrast to retrovirus, the adenoviral infection of host cells does not result in chromosomal integration because adenoviral DNA can replicate in an episomal manner without potential genotoxicity. Also, adenoviruses are structurally stable, and no genome rearrangement has been detected after extensive amplification.

Adenovirus is particularly suitable for use as a gene transfer vector because of its midsized genome, ease of manipulation, high titer, wide target-cell range and high infectivity. A person of ordinary skill in the art would be familiar with experimental methods using adenoviral vectors.

The adenovirus vector may be replication defective, or at least conditionally defective, and the nature of the adenovirus vector is not believed to be crucial to the successful practice of the invention. The adenovirus may be of any of the 42 different known serotypes or subgroups A-F and other serotypes or subgroups are envisioned. Adenovirus type 5 of subgroup C is the preferred starting material in order to obtain the conditional replication- defective adenovirus vector for use in the present invention. This is because Adenovirus type 5 is a human adenovirus about which a great deal of biochemical and genetic information is known, and it has historically been used for most constructions employing adenovirus as a vector. Adenovirus growth and manipulation is known to those of skill in the art, and exhibits broad host range in vitro and in vivo. Modified viruses, such as adenoviruses with alteration of the CAR domain, may also be used. Methods for enhancing delivery or evading an immune response, such as liposome encapsulation of the virus, are also envisioned.

The retroviruses are a group of single-stranded RNA viruses characterized by an ability to convert their RNA to double-stranded DNA in infected cells by a process of reverse- transcription (Coffin, 1990). The resulting DNA then stably integrates into cellular chromosomes as a provirus and directs synthesis of viral proteins. The integration results in the retention of the viral gene sequences in the recipient cell and its descendants. The retroviral genome contains two long terminal repeat (LTR) sequences present at the 5' and 3' ends of the viral genome. These contain strong promoter and enhancer sequences and are also required for integration in the host cell genome (Coffin, 1990).

In order to construct a retroviral vector, a nucleic acid encoding a nucleic acid or gene of interest is inserted into the viral genome in the place of certain viral sequences to produce a virus that is replication-defective. A person of ordinary skill in the art would be familiar with well-known techniques that are available to construct a retroviral vector.

Adeno-associated virus (AAV) is an attractive vector system for use in the present invention as it has a high frequency of integration and it can infect nondividing cells, thus making it useful for delivery of genes into mammalian cells in tissue culture (Muzyczka, 1992). AAV has a broad host range for infectivity (Tratschin, et al, 1984; Laughlin, et al, 1986; Lebkowski, et al, 1988; McLaughlin, et al, 1988), which means it is applicable for use with the present invention. Details concerning the generation and use of rAAV vectors are described in U.S. Patents 5,139,941 and 4,797,368, each incorporated herein by reference.

Typically, recombinant AAV (rAAV) virus is made by cotransfecting a plasmid containing the gene of interest flanked by the two AAV terminal repeats (McLaughlin et al, 1988; Samulski et al, 1989; each incorporated herein by reference) and an expression plasmid containing the wild-type AAV coding sequences without the terminal repeats, for example pIM45 (McCarty et al, 1991; incorporated herein by reference). A person of ordinary skill in the art would be familiar with techniques available to generate vectors using AAV virus.

Herpes simplex virus (HSV) has generated considerable interest in treating nervous system disorders due to its tropism for neuronal cells, but this vector also can be exploited for other tissues given its wide host range. Another factor that makes HSV an attractive vector is the size and organization of the genome. Because HSV is large, incorporation of multiple genes or expression cassettes is less problematic than in other smaller viral systems. In addition, the availability of different viral control sequences with varying performance (temporal, strength, etc.) makes it possible to control expression to a greater extent than in other systems. It also is an advantage that the virus has relatively few spliced messages, further easing genetic manipulations.

HSV also is relatively easy to manipulate and can be grown to high titers. Thus, delivery is less of a problem, both in terms of volumes needed to attain sufficient MOI and in a lessened need for repeat dosings. For a review of HSV as a gene therapy vector, see Glorioso et al (1995). A person of ordinary skill in the art would be familiar with well- known techniques for use of HSV as vectors.

Vaccinia virus vectors have been used extensively because of the ease of their construction, relatively high levels of expression obtained, wide host range and large capacity for carrying DNA. Vaccinia contains a linear, double-stranded DNA genome of about 186 kb that exhibits a marked "A-T" preference. Inverted terminal repeats of about 10.5 kb flank the genome.

Other viral vectors may be employed as constructs in the present invention. For example, vectors derived from viruses such as poxvirus may be employed. A molecularly cloned strain of Venezuelan equine encephalitis (VEE) virus has been genetically refined as a replication competent vaccine vector for the expression of heterologous viral proteins (Davis et al, 1996). Studies have demonstrated that VEE infection stimulates potent CTL responses and has been suggested that VEE may be an extremely useful vector for immunizations (Caley et al, 1997). It is contemplated in the present invention, that VEE virus may be useful in targeting dendritic cells.

A polynucleotide may be housed within a viral vector that has been engineered to express a specific binding ligand. The virus particle will thus bind specifically to the cognate receptors of the target cell and deliver the contents to the cell. A novel approach designed to allow specific targeting of retrovirus vectors was developed based on the chemical modification of a retrovirus by the chemical addition of lactose residues to the viral envelope. This modification can permit the specific infection of hepatocytes via sialoglycoprotein receptors.

Another approach to targeting of recombinant retroviruses was designed in which biotinylated antibodies against a retroviral envelope protein and against a specific cell receptor were used. The antibodies were coupled via the biotin components by using streptavidin (Roux et al, 1989). Using antibodies against major histocompatibility complex class I and class II antigens, they demonstrated the infection of a variety of human cells that bore those surface antigens with an ecotropic virus in vitro (Roux et al, 1989). 2 Nonviral Vectors

Several non-viral methods for the transfer of nucleic acids into cells also are contemplated by the present invention. These include calcium phosphate precipitation (Graham and Van Der Eb, 1973; Chen and Okayama, 1987; Rippe et al, 1990) DEAE- dextran (Gopal, 1985), electroporation (Tur-Kaspa et al, 1986; Potter et al, 1984), direct microinjection (Harland and Weintraub, 1985), DNA-loaded liposomes (Nicolau and Sene, 1982; Fraley et al, 1979) and lipofectamine-DNA complexes, polyamino acids, cell sonication (Fechheimer et al, 1987), gene bombardment using high velocity microprojectiles (Yang et al, 1990), polycations (Bousssif et al, 1995) and receptor-mediated transfection (Wu and Wu, 1987; Wu and Wu, 1988). Some of these techniques may be successfully adapted for in vivo or ex vivo use. A person of ordinary skill in the art would be familiar with the techniques pertaining to use of nonviral vectors, and would understand that other types of nonviral vectors than those disclosed herein are contemplated by the present invention.

In a further embodiment of the invention, the expression cassette may be entrapped in a liposome or lipid formulation. Liposomes are vesicular structures characterized by a phospholipid bilayer membrane and an inner aqueous medium. Multilamellar liposomes have multiple lipid layers separated by aqueous medium. Also contemplated is a gene construct complexed with Lipofectamine (Gibco BRL). One of ordinary skill in the art would be familiar with techniques utilizing liposomes and lipid formulations. Lipid based non-viral formulations provide an alternative to adenoviral gene therapies.

Although many cell culture studies have documented lipid based non-viral gene transfer, systemic gene delivery via lipid based formulations has been limited. A major limitation of non- viral lipid based gene delivery is the toxicity of the cationic lipids that comprise the non- viral delivery vehicle. The in vivo toxicity of liposomes partially explains the discrepancy between in vitro and in vivo gene transfer results. Another factor contributing to this contradictory data is the difference in liposome stability in the presence and absence of serum proteins. The interaction between liposomes and serum proteins has a dramatic impact on the stability characteristics of liposomes (Yang and Huang, 1997). Cationic liposomes attract and bind negatively charged serum proteins. Liposomes coated by serum proteins are either dissolved or taken up by macrophages leading to their removal from circulation. Current in vivo liposomal delivery methods use subcutaneous, intradermal, intratumoral, or intracranial injection to avoid the toxicity and stability problems associated with cationic lipids in the circulation. The interaction of liposomes and plasma proteins is responsible for the disparity between the efficiency of in vitro (Feigner et al, 1987) and in vivo gene transfer (Zhu et al, 1993; Solodin et al, 1995; Thierry et al, 1995; Tsukamoto et al, 1995; Aksentijevich et al, 1996).

The production of lipid formulations often is accomplished by sonication or serial extrusion of liposomal mixtures after (I) reverse phase evaporation (II) dehydration- rehydration (III) detergent dialysis and (IV) thin film hydration. Once manufactured, lipid structures can be used to encapsulate compounds that are toxic (chemotherapeutics) or labile

(nucleic acids) when in circulation. Liposomal encapsulation has resulted in a lower toxicity and a longer serum half-life for such compounds (Gabizon et al, 1990). Numerous disease treatments are using lipid based gene transfer strategies to enhance conventional or establish novel therapies, in particular therapies for treating hyperproliferative diseases.

IV. COMBINATION THERAPY

It is an aspect of this invention that the claimed methods for treating cells in a subject can be used in combination with another agent or therapy method, hi certain embodiments, the disease is cancer, the other agent or therapy is another anti-cancer agent or anti-cancer therapy. Treatment with the claimed dual therapeutic agent may precede or follow the other therapy method by intervals ranging from minutes to weeks. In embodiments where another agent is administered, one would generally ensure that a significant period of time did not expire between the time of each delivery, such that the agents would still be able to exert an advantageously combined effect on the cell. For example, it is contemplated that one may administer two, three, four or more doses of one agent substantially simultaneously {i.e., within less than about a minute) with the dual therapeutic agents of the present invention. In other aspects, a therapeutic agent or method may be administered within about 1 minute to about 48 hours or more prior to and/or after administering a dual therapeutic agent or agents of the present invention, or prior to and/or after any amount of time not set forth herein. In certain other embodiments, a dual therapeutic agent of the present invention may be administered within of from about 1 day to about 21 days prior to and/or after administering another therapeutic modality, such as surgery or gene therapy. In some situations, it may be desirable to extend the time period for treatment significantly, however, where several weeks {e.g., about 1 to 8 weeks or more) lapse between the respective administrations. Various combinations may be employed, the claimed agent for dual chemotherapy and radiation therapy is derivative is "A" and the secondary agent , which can be any other therapeutic agent or method, is "B":

A/B/A B/A/B B/B/A AJAJB A/B/B BIAJA A/B/B/B B/A/B/B B/B/B/A B/B/A/B A/A/B/B A/B/A/B A/B/B/A B/B/A/A

B/A/B/A B/A/A/B A/A/A/B B/A/A/A A/B/A/A A/A/B/A

Administration of the dual therapeutic agents of the present invention to a patient will follow general protocols for the administration of chemotherapeutics, taking into account the toxicity, if any, of these agents. It is expected that the treatment cycles would be repeated as necessary. It also is contemplated that various standard therapies, as well as surgical intervention, may be applied in combination with the therapeutic. These therapies include but are not limited to additional drug therapy, chemotherapy, additional radiotherapy, immunotherapy, gene therapy and surgery.

A. Chemotherapy

Cancer therapies also include a variety of combination therapies with both chemical and radiation based treatments. Chemotherapies include, but are not limited to, for example, cisplatin (CDDP), carboplatin, procarbazine, mechlorethamine, cyclophosphamide, camptothecin, ifosfamide, melphalan, chlorambucil, busulfan, nitrosurea, dactinomycin, daunorubicin, doxorubicin, bleomycin, plicomycin, mitomycin, etoposide (VP 16), tamoxifen, raloxifene, estrogen receptor binding agents, taxol, gemcitabien, navelbine, farnesyl-protein tansferase inhibitors, transplatinum, 5-fluorouracil, vincristin, vinblastin and methotrexate, or any analog or derivative variant of the foregoing.

B. Radiotherapy Other factors that cause DNA damage and have been used extensively include what are commonly known as γ-rays, X-rays, and/or the directed delivery of radioisotopes to tumor cells. Other forms of DNA damaging factors are also contemplated such as microwaves and UV-irradiation. It is most likely that all of these factors effect a broad range of damage on DNA, on the precursors of DNA, on the replication and repair of DNA, and on the assembly and maintenance of chromosomes. Dosage ranges for X-rays range from daily doses of 50 to 200 roentgens for prolonged periods of time (3 to 4 wk), to single doses of 2000 to 6000 roentgens. Dosage ranges for radioisotopes vary widely, and depend on the half-life of the isotope, the strength and type of radiation emitted, and the uptake by the neoplastic cells. The terms "contacted" and "exposed," when applied to a cell, are used herein to describe the process by which a therapeutic construct and a chemo therapeutic or radiotherapeutic agent are delivered to a target cell or are placed in direct juxtaposition with the target cell. To achieve cell killing or stasis, both agents are delivered to a cell in a combined amount effective to kill the cell or prevent it from dividing.

C. Immunotherapy

Irnmunotherapeutics, generally, rely on the use of immune modulators, immune effector cells and molecules to cure or palliate disease. In certain embodiments, immune modulators, immune effector cells and molecules target and destroy cancer cells. The immune effector may be, for example, an antibody specific for some marker on the surface of a tumor cell. The antibody alone may serve as an effector of therapy or it may recruit other cells to actually effect cell killing. The antibody also may be conjugated to a drug or toxin (chemotherapeutic, radionucleotide, ricin A chain, cholera toxin, pertussis toxin, etc.) and serve merely as a targeting agent. Alternatively, the effector may be a lymphocyte carrying a surface molecule that interacts, either directly or indirectly, with a tumor cell target. Various effector cells include cytotoxic T cells and NK cells.

Immunotherapy, thus, could be used as part of a combined therapy, in conjunction with gene therapy. The general approach for combined therapy is discussed below. Generally, the tumor cell must bear some marker that is amenable to targeting, i.e., is not present on the majority of other cells. Many tumor markers exist and any of these may be suitable for targeting in the context of the present invention. Common tumor markers include carcino embryonic antigen, prostate specific antigen, urinary tumor associated antigen, fetal antigen, tyrosinase (p97), gp68, TAG-72, HMFG, Sialyl Lewis Antigen, MucA, MucB, PLAP, estrogen receptor, laminin receptor, erb B and pi 55.

D. Genes

In yet another embodiment, the secondary treatment is a second gene therapy in which a therapeutic polynucleotide is administered before, after, or at the same time as the nucleic acid composition of the present invention. Delivery of the dual therapeutic agent in conjunction with a vector encoding a gene product will have a combined therapeutic effect such as an anti-hyperproliferative effect on target tissues.

E. Surgery

Approximately 60% of persons with cancer will undergo surgery of some type, which includes preventative, diagnostic or staging, curative and palliative surgery. Curative surgery is a cancer treatment that may be used in conjunction with other therapies, such as the treatment of the present invention, chemotherapy, radiotherapy, hormonal therapy, gene therapy, immunotherapy and/or alternative therapies. Curative surgery includes resection in which all or part of cancerous tissue is physically removed, excised, and/or destroyed. Tumor resection refers to physical removal of at least part of a tumor. In addition to tumor resection, treatment by surgery includes laser surgery, cryosurgery, electrosurgery, and miscopically controlled surgery (Mohs' surgery). It is further contemplated that the present invention may be used in conjunction with removal of superficial cancers, precancers, or incidental amounts of normal tissue.

V. PHARMACEUTICAL PREPARATIONS

Certain embodiments of the present invention involve introducing a pharmaceutically acceptable dose of a nucleic acid encoding a reporter detectable in vivo using non-invasive methods operatively coupled to a tissue-selective promoter to a subject. Pharmaceutical compositions of the present invention comprise a therapeutically or diagnostically effective amount of a nucleic acid of the present invention. The phrases "pharmaceutical or pharmacologically acceptable" or "therapeutically effective" or "diagnostically effective" refers to molecular entities and compositions that do not produce an unacceptable adverse, allergic or other untoward reaction when administered to an animal, such as, for example, a human, as appropriate. The preparation of therapeutically effective or diagnostically effective compositions will be known to those of skill in the art in light of the present disclosure, as exemplified by Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, incorporated herein by reference. Moreover, for animal (e.g., human) administration, it will be understood that preparations should meet sterility, pyrogenicity, general safety and purity standards as required by FDA Office of Biological Standards.

As used herein, "a composition comprising a therapeutically effective amount" or "a composition comprising a diagnostically effective amount" includes any and all solvents, dispersion media, coatings, surfactants, antioxidants, preservatives {e.g., antibacterial agents, antifungal agents), isotonic agents, absorption delaying agents, salts, preservatives, drugs, drug stabilizers, gels, binders, excipients, disintegration agents, lubricants, sweetening agents, flavoring agents, dyes, such like materials and combinations thereof, as would be known to one of ordinary skill in the art. Except insofar as any conventional carrier is incompatible with the active ingredient, its use in the present compositions is contemplated.

The compositions of the present invention may comprise different types of carriers depending on whether it is to be administered in solid, liquid or aerosol form, and whether it need to be sterile for such routes of administration as injection. The dual imaging agents and dual therapeutic agents of the present invention can be administered intravenously, intradermally, intraarterially, intraperitoneally, intralesionally, intracranially, intraarticularly, intraprostaticaly, intrapleurally, intratracheally, intranasally, intravitreally, intravaginally, intrarectally, topically, intratumorally, intramuscularly, intraperitoneally, subcutaneously, subconjunctival, intravesicularlly, mucosally, intrapericardially, intraumbilically, intraocularally, orally, topically, locally, injection, infusion, continuous infusion, localized perfusion bathing target cells directly, via a catheter, via a lavage, in lipid compositions (e.g., liposomes), or by other method or any combination of the forgoing as would be known to one of ordinary skill in the art. The actual required amount of a composition of the present invention administered to a patient can be determined by physical and physiological factors such as body weight, severity of condition, the type of disease being treated, previous or concurrent therapeutic interventions, idiopathy of the patient and on the route of administration. The practitioner responsible for administration will, in any event, determine the concentration of active ingredient(s) in a composition and appropriate dose(s) for the individual subject.

In certain embodiments, pharmaceutical compositions may comprise, for example, at least about 0.1% of a dual imaging agent or a dual therapeutic agent. In other embodiments, the active compound may comprise between about 2% to about 75% of the weight of the unit, or between about 25% to about 60%, for example, and any range derivable therein. In other non-limiting examples, a dose may also comprise from about 0.1 mg/kg/body weight to about 1000 mg/kg/body weight or any amount within this range, or any amount greater than 1000 mg/kg/body weight per administration

In any case, the composition may comprise various antioxidants to retard oxidation of one or more component. Additionally, the prevention of the action of microorganisms can be brought about by preservatives such as various antibacterial and antifungal agents, including, but not limited to parabens (e.g., methylparabens, propylparabens), chlorobutanol, phenol, sorbic acid, thimerosal or combinations thereof.

The dual imaging agents and dual therapeutic agents of the present invention may be formulated into a composition in a free base, free acid, neutral or salt form. Pharmaceutically acceptable salts include the salts formed with the free carboxyl groups derived from inorganic bases such as for example, sodium, potassium, ammonium, calcium or ferric hydroxides; or such organic bases as isopropylamine, trimethylamine, histidine or procaine.

In embodiments where the composition is in a liquid form, a carrier can be a solvent or dispersion medium comprising, but not limited to, water, ethanol, polyol (e.g., glycerol, propylene glycol, liquid polyethylene glycol, etc.), lipids {e.g., triglycerides, vegetable oils, liposomes) and combinations thereof. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin; by the maintenance of the required particle size by dispersion in carriers such as, for example liquid polyol or lipids; by the use of surfactants such as, for example hydroxypropylcellulose; or combinations thereof such methods. In many cases, it will be preferable to include isotonic agents, such as, for example, sugars, sodium chloride or combinations thereof.

Sterile injectable solutions are prepared by incorporating the diagnostic or therapeutic agent in the required amount of the appropriate solvent with various amounts of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and/or the other ingredients. In the case of sterile powders for the preparation of sterile injectable solutions, suspensions or emulsion, the preferred methods of preparation are vacuum-drying or freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered liquid medium thereof. The liquid medium should be suitably buffered if necessary and the liquid diluent first rendered isotonic prior to injection with sufficient saline or glucose. The preparation of highly concentrated compositions for direct injection is also contemplated, where the use of DMSO as solvent is envisioned to result in extremely rapid penetration, delivering high concentrations of the active agents to a small area.

The composition must be stable under the conditions of manufacture and storage, and preserved against the contaminating action of microorganisms, such as bacteria and fungi. It will be appreciated that endotoxin contamination should be kept minimally at a safe level, for example, less that 0.5 ng/mg protein.

In particular embodiments, prolonged absorption of an injectable composition can be brought about by the use in the compositions of agents delaying absorption, such as, for example, aluminum monostearate, gelatin or combinations thereof.

EXAMPLES The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

EXAMPLE 1

Non-Invasively Quantifying Exogenous Gene Expression in Tumors by Functional and

Anatomic Imaging

The purpose of this study was to assess whether a combination of functional (planar and SPECT imaging) and anatomic (MR) techniques can be used to non-invasively quantify tumor expression of a somatostatin receptor type 2 (SSTR2) gene chimera in vivo.

Materials and Methods Plasmid

As described in Kundra et al., 2002, the entire contents of which is specifically incorporated by reference herein for this section as well as for the entire patent application, the full-length human SSTR2A was inserted into the pDisplay vector (Invitrogen, Carlsbad,

CA), downstream of the membrane localization sequence and the hemagglutinin A (HA) epitope tag sequence.

Cell Line Production and Characterization

HTl 080 cells (human fibrosarcoma, ATCC, Rockville, MD) were grown in Dulbecco's Modified Eagle Medium (DMEM) containing Ix glutamine, penicillin, and streptomycin (GPS) and 10% fetal bovine serum (FBS). For transfection, 1 μg DNA was added with lipofectin 2000 (Invitrogen, Carlsbad, CA) to 1 x 10⁵ cells according to the manufacturer's instructions. After 5 h, the lipofectin-DNA solution was removed, and the cells were incubated in growth medium. After G418 selection, single colonies were isolated. Prospective colonies were assayed for expression by the enzyme-linked immunosorbent assay (ELISA) and then individual clones were assessed for expression quantitatively. ELISA and receptor binding studies were performed as described previously (Kundra et al, 2002). Western Blot Analysis

For cells, confluent 6-well dishes, were exposed to Triton X-IOO-SDS lysis solution (0.1% sodium dodecyl sulfate [SDS], 1% Triton X-100, 0.1 mol/L Tris [pH 8], 0.14 mol/L sodium chloride, 0.025% sodium azide, and 0.18% Complete protease inhibitor [Roche]) for 1 h at 4°C. After a 30-min centrifugation at 14,000 x g, the supernatant was collected. For tumors, samples were washed with PBS, and homogenized with 10 strokes in TritonX-100- SDS lysis solution. After a 15 minutes centrifugation at 14,000 x g, the supernatant was collected. Protein concentration was determined using the Bradford method (Bio-Rad Laboratories, Hercules, CA). Twenty micrograms of cell protein or fifty micrograms of tissue protein were loaded per lane on 7% SDS gels. The sample was then transferred to nitrocellulose using a semidry apparatus (Fisher, Atlanta, GA). Equal transfer was confirmed by Ponceau-S staining. Next, the membrane was blocked and exposed to 50 mU/mL HRP- rat-anti-HA antibody overnight at 4°C or 1 h at room temperature. After four 10-min washes with PBS, the membrane was covered with a chemiluminescent HRP substrate (Perkin Elmer Life Science, Boston, MA) for filming.

Immunohistochemistry

Paraffin embedded tumor tissue was probed with a 1 :1000 dilution of a mouse anti-

HA antibody (Babco, Richmond, CA), washed and then stained with an HRP-conjugated antimouse secondary antibody. The tissue was counterstained with Giemsa. The presence of a brown reaction product at the periphery of the cells was considered a positive reaction for the fusion.

Biodistribution and Imaging All animal experiments were approved by the institutional animal care and use committee. In 9 nude mice (approximately 8 weeks old and 25 g), subcutaneous injection of 5 x 10⁶ cells produced palpable tumors after 1 week. Each mouse received three inoculations of tumors: right thigh, cells transfected with vector; right and left shoulder, clone 309 and 301 respectively, which express different levels of the same gene chimera. Next, 6 of the 9 mice were randomly selected for tail vein injection with 13 MBq (350 μCi) of ¹¹¹In-OCt (Mallinckrodt, St. Louis, Mo).

24 hours later, anesthetized animals were imaged with a 4.7 T small animal MR (Bruker, Billerica, MA) using a T2-FSE sequence (TE 4120 msec, TR 72 msec, 4 Nex, field of view 3.5 cm, slice thickness 1 mm with 0.3 mm skip, matrix 256 x 256, resolution 136 microns). Tumor measurements were performed using Image J software (National Institutes of Health, USA). In each image containing a tumor, the periphery of the mass was traced and the area of the drawn region was calculated. The areas were then multiplied by the slice thickness plus skip to obtain the volume of each slice containing the object of interest. Each slice volume was then added. However, to control for volume averaging, only one half of the volume of the most superior and one half of the volume of the most inferior images containing the object of interest were added. Assuming a tissue density of 1 g/ml, to derive weight, the volume of the object of interest in mm³ was multiplied by .001 g of tissue/mm³. The same process was used to trace and calculate the weight of necrotic/hemorrhagic material, identified on MR as areas of increased and decreased signal compared to the tumor and containing fluid-fluid/debris levels. The weight of the necrotic/hemorrhagic material was then subtracted from the weight of the mass to calculate the corrected weight.

Next the mice underwent planar imaging for 10 minutes using a γ-camera (mCAM, Siemens Medical Solutions, Hoffman Estates, IL) fitted with a medium-energy parallel hole collimator. No attenuation correction was used. For SPECT, fifteen minute acquisitions were obtained. Imaging consisted of 120 views (7.5 sec/view, 128x128, 2.4 mm/pixel) over a 360 rotation of a fixed 1/15 rpm rotational device attached to the front of the collimator. Each animal was spun in the rotational device. For SPECT reconstruction, Butterworth 0.6 Nyquist, 10^th order filtered-backprojection was performed. In planar and SPECT images, region of interest total count measurements were normalized to the number of pixels in the region of interest (ROI) to obtain average counts/pixel. For the planar images, these values were subtracted from those obtained from the left thigh, which did not have a tumor. For both techniques, the values were then converted to counts per minute using an equation derived from phantoms containing relevant amounts of activity. Phantoms consisted of 1.5 ml Eppendorf tubes containing 500 μl of different amounts of ¹ ^n-chloride (93-0.3 μCi). The fluid in each tube was similar in size to a tumor in a mouse. Because each tumor was only seen in one to three SPECT images, the image with the greatest amount of counts was selected. This same methodology was used with the phantoms. The derived counts per minute values were then normalized to injected dose in counts per minute to obtain percent- inj ected dose (%ID) .

Afterwards, the mice were sacrificed and each organ and tumor dissected, weighed, and associated radioactivity determined via a gamma-counter. Simultaneously, the remaining 3 of 9 mice were sacrificed and tumors were removed for Western blotting and immunohistochemistry to assess expression of the gene chimera. Influence of Radioactivity

For assessing the influence of the amount of radioactivity on image representation, 1.5 ml Eppendorf tubes were filled with 0.5 ml of phosphate buffered saline containing serial 1 :1 dilutions of ⁿ ^-chloride from 93 to .03 μCi. Planar imaging was performed as described above. Using a variety of background and saturation display levels, the size of the phantom on the image was visually compared to the size of the actual phantom.

Statistical Analysis Wilcoxon Rank Sum test (one sided) was used to compare expression in vitro or uptake among tumors that differed in expression in vivo. Linear regression was used to analyze correlations. The analyses were performed using Excel (2000, Microsoft, Bellevue, Washington). Using SAS (2001, version 8.02, SAS Institute, Cary, N.C.), the Kruskal Wallis test was utilized to compare trend among tumors that differed in expression. For all tests, p<.05 was considered significant.

RESULTS

Expression

Using an antibody to the HA domain of the fusion protein, expression was confirmed in whole cells by ELISA (normalized for cell number) and in cell lysates by Western blot analysis (normalized for protein). In a quantitative ELISA of clonal lines transfected with the same SSTR2A gene chimera (FIG. IA), clone 309 reacted more than clone 301. In comparison, no reaction was seen in cells transfected with only vector. As seen on the

Western blot (FIG. IB), a distinct band is observed in all lanes representing cells transfected with the SSTR2A chimeric gene but not in lanes representing cells transfected with vector only. The band is more intense in the lane marked clone 309 than marked 301, implying greater expression in clone 309. No expression is seen in cells transfected with only vector.

To confirm proper function of the SSTR2A portion of the fusion protein, receptor- binding assays were performed. For the assay, a saturating dosing of 10^"7 M octreotide was used (Reisine et al, 1993; Raynor et ah, 1993). Because both HTl 080 cell clones express the same fusion protein, any difference in binding is due to the amount of expression. As seen in FIG. 1C, binding to ¹¹¹In-OCt is greater for clone 309 than for clone 301. Unlabeled somatostatin competes with the labeled analogue, confirming specificity. No specific binding is seen in cells transfected with vector only. The receptor-binding data correspond with the ELISA and Western blot analysis. Thus, expression levels based on the anti-HA antibody corroborate receptor-binding data based on ^{π 1}In-OCt. These data demonstrate that a greater amount of fusion protein per cell is present in clone 309 than in clone 301 and that the HA and SSTR2A domains of the fusion proteins are functional.

Biodistribution

FIG. 2 demonstrates ex vivo biodistribution analysis of ¹¹¹In-OCt 24 hours after tail vein injection into six nude mice. Each mouse bears three subcutaneous tumors derived from clones 309, 301, or vector-transfected cells. Unbound ¹¹¹In-Od: is eliminated through the kidneys and liver and there was variability in the extent of each route used among the mice. Comparing findings in excised tumors, there is a statistically significant difference in the biodistribution parameter of %ID/g among tumors originating from vector or the gene chimera-transfected cells (vector versus 301, p<.05, n=6; vector versus 309, p<.05, n=6) and between clone 301 and 309 (p < 0.05, n=6). Thus, tumors expressing different levels of fusion protein are distinguished by the invasive biodistribution methods.

Functional Imaging

Tumor-bearing nude mice were imaged 24 hours after tail vein injection of ¹¹¹In-OCt, but before sacrificing the animals for the biodistribution analysis of FIG. 2. A planar image of a representative mouse (FIG. 3A) demonstrates that the tumor derived from clone 309, which expresses more fusion protein, is better visualized than the tumor derived from clone 301. Tumor derived from cells transfected with vector appears similar to background. To improve localization, tomographic methods were applied. SPECT imaging of gene expression in a representative mouse (FIG. 3B, FIG. 3C, FIG. 3D) demonstrates the tumors expressing the fusion protein, whereas tumor derived from vector transfected cells appears similar to background. For both planar (r=.94, ρ< .05, n=18) (FIG. 3E) and SPECT (r=.9O, p< .05, n=18) (FIG. 3F) imaging, uptake derived from ROI analysis of the images correlated with radioactivity associated with excised tumors (coefficients.8, S.E.=.2; coefficients.3, S.E.^ respectively). Among the imaging methods, the correlation coefficient between the planar and SPECT techniques was 0.96 (ρ<.05, n=18, coefficients .3, S.E.=.l). However, the size of the object on the functional gamma camera image may not reflect the true size of the object.

The planar image in FIG. 3 G demonstrates that although all of the phantoms are of the same size, the apparent size of each phantom in the image increases with increasing amounts of radioactivity. In addition, there is apparent heterogeneity in the color representation of each individual object although the phantoms have a uniform distribution of radiopharmaceutical .

Anatomic Imaging

Before sacrificing the animals for the biodistribution analysis of FIG. 2, the mice were also imaged by MR. The pelvis image of a representative mouse (FIG. 4A) shows that the tumor has intermediate signal and demonstrates excellent contrast with adjacent structures. Weight derived from volume assessment of the entire mass on the tomographic MR images (FIG. 4B) correlates with the weight of the excised tumors (r=.98, p< 0.05, 11=18, coefficients.03, S.E.=.O6). Significant correlation was not seen between weight of the excised tumor and weight derived from volume assessment of the mass on the SPECT images (r=0, p>.05, n=18) or area derived from planar images (r=0, p>.05, n=18).

In some tumors, high T2 signal greater than that within the soft tissue of the mass was seen (FIG. 4C). In addition, layering of low T2 signal was noted within these regions of high signal, consistent with fluid-fluid or fluid-debris levels due to hemorrhage/necrosis. Because these areas are not expected to contain significant numbers of live cells expressing the fusion protein, they were subtracted for calculating corrected tumor weight.

Non-invasive Versus Invasive Assessment of Uptake

The association of the in vitro and in vivo findings was further examined by regression analysis. The biodistribution parameter (FIG. 5) of % injected dose/gram (% LD Jg), evaluated using excised tumors, correlates with the non-invasive, image-derived values using planar (r=.9O, p<.05, n=18, coefficients.8, S.E.= 2) or SPECT (r=.87, p<.05, n=18, coefficients .5, S.E.=.2) techniques for determining uptake and MR for the weight of the entire tumor.

The biodistribution of 'in-oct among excised tumors was distinguishable using the parameter % I.D./g (FIG. 6A). Normalizing instead to corrected weight derived from MR still allows separation of biodistribution among tumors (FIG. 6B). This was also found when completely image based parameters were derived using planar (FIG. 6C) or SPECT (FIG. 6D) techniques for assessing uptake and MR for corrected weight. By each method employed in FIG. 6A, FIG. 6B, FIG. 6C, or FIG. 6D, using either excised tumors or the in vivo image derived parameters, tumors originating from clone 309 had statistically significantly greater expression than those from clone 301 (ρ<.05, n=6), and both of these demonstrated greater ¹¹¹In-OCt biodistribution than tumors originating from cells transfected with vector (vector versus 301, p<.05, n=6; vector versus 309, p<.05, n=6). The Kruskal-Wallis test also indicated a difference in expression using any of the methods employed in FIG. 6A, FIG. 6B, FIG. 6C, or FIG. 6D (p<.05).

Ex-vivo expression analysis

To further validate expression ex-vivo, on the same day as the imaging, the three additional tumor-bearing mice were sacrificed. Portions of the excised tumors were analyzed by Western blotting using an antibody to the HA tag portion of the fusion protein. Expression is greater in tumors derived from clone 309 than those from clone 301 (FIG. 7A). No band is seen in tumors derived from cells transfected with vector only. Immunohistochemical analysis (FIG. 7B) confirms that expression in tumors derived from clone 309 is greater than in tumors derived from clone 301. As expected, staining is seen at the periphery of the cells, which is consistent with cell membrane localization of the fusion protein. Background staining is seen in tumors derived from cells transfected with vector. These data of varying degrees of expression among the clones and their subsequently derived tumors are consistent with the in vitro and in vivo findings.

These results demonstrate that a combination of non-invasive functional and anatomic imaging can be used in vivo to quantify gene transfer in tumors. Further, these data demonstrate that non-invasive imaging criteria can substitute for invasive methods for following gene expression in tumors. In addition, morphologic data can be used to identify and exclude regions of the tumor that cannot contribute to gene expression. These data demonstrate that these measurements can be obtained in small animals such as mice. Combining functional and anatomic imaging techniques for non-invasive determination of biodistribution should also be applicable to other functional methodologies such as PET and optical imaging as well as anatomic modalities such as CT. Because most of these instruments are available for patient evaluation, these techniques should find clinical utility.

EXAMPLE 2 Development of Tumor-Selective hTMC Plasmid Vector for Therapeutic Gene

Expression and Molecular Imaging

One of the major obstacles to successful cancer gene therapy is the lack of an effective delivery system that can be specifically targeted to tumors. One means for tumor-targeted transgene expression is to control gene expression via tumor-specific promoters. Human telomerase reverse transcriptase (hTERT) is the catalytic subunit of telomerase, which is highly active in immortalized cells and in more than 85% of human cancers but is quiescent in most normal somatic cells (Shay and Bacchetti, 1997; Shay et al, 2001). The hTERT promoter has been cloned and shown to be capable of selectively promoting transgene expression in tumors but not in normal cells (Nakayama et al, 1998). However, the transcription-promoting strength of the hTERT promoter, like most other intrinsic mammalian promoters, is much weaker than commonly used viral promoters such as the CMV and the SV40 early promoters. Consequently, its use for cancer gene therapy is hampered by the problem of low transgene expression (Nakayama et al, 1998; Gu et al, 2000). To circumvent these problems, a novel chimeric promoter, hTERT-mini-CMV

(hTMC), has been engineered by optimally fusing essential hTERT promoter sequences with minimal CMV promoter elements. Using this novel promoter, both high tumor-specificity and high transgene expression in vitro and in vivo have been achieved, as demonstrated here using an hTMC-EGFP reporter system in vivo (FIG. 8). Human N417 lung cancer tumor-bearing mice were intravenously injected with various EGFP-nanoparticles, in which EGFP expression is driven by either original CMV or hTERT promoters versus the hTMC promoter. Forty-eight hours after injection, the mice were killed and organs were collected and frozen immediately. Fresh frozen tissue sections were examined under a fluorescence microscope for EGFP expression. A high level of EGFP expression was detected only in the tumor cells in animals treated with hTMC-EGFP but not in any other normal tissues (FIG. 8, panel hTMC). By contrast, very weak tumor-specific EGFP expression was detected in animals treated with TERT-EGFP, and non-selective expression was detected in tumor and normal tissues in animals treated by the CMV-EGFP construct.

EXAMPLE 3

Gamma-Camera Imaging of Gene Transfer In vivo

Many molecular imaging methods for monitoring gene transfer in vivo rely on the sensitivity of gamma-camera, SPECT, or PET imaging for the detection of intravenously injected radiolabeled compounds localized to the products of transferred genes, but most neither directly detect the gene of interest nor exploit radiopharmaceuticals that have FDA approval for total-body use (Yamada et al, 1992; Kundra et al, 2002). The radiopharmaceutical, ^mIn-Octreotide, has successfully detected the cell surface membrane localization of SSTR2A products in HT1080 cells. Cell membrane localization of the product in HTl 080 cells transfected with HA-SSTR2A-expressing plasmids (pHA-SSTA2A) was confirmed by immunofluorescence analysis with an anti-HA antibody (FIG. 9A). Mice bearing HT1080 tumors produced by pHA-SSTR2A-transfectants were injected with ¹¹¹In- Octreotide for biodistribution and imaging studies by gamma-camera (FIG. 9B). SSTR2A expression was also shown to be significantly correlated with both the receptor binding and with the ^u ^-Octreotide radiotracer uptake by tumors (FIG. 9D) (Kundra et al, 2002). This approach further demonstrated feasibility for clinical use because gene transfer could be detected in tumor-bearing animals with ⁿ ^-Octreotide at doses similar to those already used in humans. A novel tumor-specific dual reporter and therapeutic gene expression plasmid vector for molecular imaging has been developed (FIG. 10). hi this vector (pLJ290), the backbone of the plasmid vector pLJ143/FUSl was used (FIG. 10A), which has been approved by the FDA for a Phase I clinical trial. The original vector was modified by inserting an improved Internal Ribosome Entry Site (IRES) between the reporter gene SSTR2A and therapeutic tumor suppressor gene FUSl (FIG. 10C). This allows simultaneous expression of both genes separately, under the control of the same promoter and with the same BGH poly (A) signal sequence. The co-expression and distinguished subcellular localizations of both the SSTR2A gene (FIG. 9D, Green, cell surface membrane) and the FUSl gene (FIG. 9D, Red, mitochondria, ER, and peri-nuclear membrane locations) are demonstrated by immunofluorescence imaging analysis. The pLJ290 vector was engineered further by replacing the CMV promoter with the hTMC promoter (FIG. 10D, pLJ294), which will allow tumor- specific expression of both the SSTR2A and the FUSl genes. These novel therapeutic vectors combined with noninvasive and sensitive gamma-camera imaging with radiopharmaceutical ^{u l}In-Octreotide will allow for efficient monitoring of the distribution, clearance, expression, and efficacy of the SSTi^^ -Frøi-nanoparticle-mediated gene transfer in mice and patients.

EXAMPLE 4

A Novel Synthetic hTERT-Mini-CMV Chimera Promoter-Driven Tumor-Selective and High-Efficiency Expression of Transgene for Systemic Cancer

Gene Therapy

One of the major obstacles to a successful cancer gene therapy is the lack of an effective systemic delivery system that can be specifically targeted to primary and metastatic tumors. The hTERT promoter has been cloned and shown to be capable of targeting transgene expression in tumors but not in normal cells. However, the weak transcription- promoting strength of hTERT promoter, like most other intrinsic mammalian promoters, has hampered its direct use for cancer gene therapy. To circumvent these problems, a novel chimera liTERT-mini-CMV (hTMC) promoter has been developed that was engineered by optimally fusing essential hTERT regulatory sequence with minimal CMV promoter elements. Various human cancers and normal cells were transfected with various EGFP- constructs, in which EGFP expression is driven by either the original CMV and hTERT promoters or by the hTMC promoter in vitro. Expression of EGFP in the transfectants was visualized by a fluorescence imaging (FI) under a fluorescence microscope and the population of EGFP -positive cells and fluorescence intensity were quantified by FACS analysis. A high level of EGFP expression driven by the hTMC promoter was detected in all tumor cells but not in normal cells. While a similar tumor-selectivity of EGFP expression driven by the hTERT promoter could be seen, the level of expression was several hundred-fold lower than that driven by hTMC promoter under the same transfection efficiency. The effectiveness of the hTMC promoter was also evaluated in vivo by systemic injection of DOTAP xholesterol- complexed hTMC-isGFP-nanoparticles into nude mice that bear intrathoracic human N417 lung tumor xenografts. Consistently, a high level of EGFP expression could be detected only in the tumor cells in animals treated with hTMC-EGFP but not in any other normal tissues. Furthermore, the above N417 tumor mouse model was used to evaluate the therapeutic efficacy of systemic treatment with a novel hTMC-Frøi-nanoparticle by non-invasive and quantitative MR imaging analysis. A significant inhibition (PO.001) of tumor growth was detected in animals treated by hTMC-Frøi-nanoparticles in less than 2 weeks of treatment compared to those untreated or treated by hTMC-EGFP-nanoparticles, as demonstrated by MR imaging and volume analysis. Induction of apoptosis was also detected in tumor cells but not in surrounding normal lung or other normal tissues in mice treated by hTMC-FUSl- nanoparticles, as shown by an in situ cell death analysis with TUNEL staining in frozen tissue samples. These results clearly demonstrate the capability of using the hTMC promoter to achieve both the high tumor-specificity and high-efficiency therapeutic gene expression in vitro and in vivo and implicate the translational applications of using the systemic administration of therapeutic hTMC-nanoparticle for tumor-targeted molecular cancer therapy. EXAMPLE 5

Development of Novel hTERT Promoter Chimeras and their use in Imaging In vitro and

In vivo

A novel construct for amplified hTERT-mediated expression of HA-S STR2 was developed. The construct, schematically depicted in FIG. 11 , includes the hTERT promoter, gal4/VP16, a gal4 binding site, and a gene chimera consisting of the hemrnaglutinin A tag and somatostatin receptor type 2 A (HA-S STR2 A) for amplified hTERT-mediated expression of HA-SSTR2A. Further, a similar novel construct wherein the SSTR2A has a C-terminal deletion has also been developed. Studies were then conducted to evaluate the usefulness of these constructs for in vitro imaging. HTl 080 cells and IMR90 cells were obtained from the American Type Culture Collection. A telomerase assay of extracts of HTl 080 and IMR90 cells was then performed according to the method of (Kim et al. 1994). With HT1080 cells, the 6 base pair repeat ladder indicated telomerase activity. The results are shown in FIG. 12. As a negative control, the HTl 080 cell extract was treated with RNAse (FIG. 12), which demonstrated no telomerase activity. Further, extract of IMR90 cells demonstrated, as expected, no telomerase activity.

HTl 080 cells and IMR90 cells were then infected with an adenovirus that included the hTERT promoter and gal4VP16 amplification system. Cells were plated for immunofluorescence and ELISA, which utilized an antibody targeting the HA domain of the HA-SSTR2A fusion protein.

Cell membrane subcellular localization of the expressed HA-S STR2 fusion protein was noted using Immunofluorescence. Representative results are shown in FIG. 13. Relatively increased expression of the expressed HA-S STR2 fusion protein was seen in individual fibrosarcoma cells, HTl 080 (FIG. 13A), which express telomerase, than in human fibroblasts, IMR-90(FIG. 13B), which do not express telomerase. Infection with an adenovirus containing a human telomerase promoter and gal4VP16 amplification system resulted in greater expression of HA-S STR2 A/infected cell in cells that express telomerase (HTl 080, human fibrosarcoma) than in cells that do not express telomerase (IMR-90, human fibroblasts) (FIG. 14). FIG. 15 demonstrated schematically a plasmid map showing that driven by an amplified hTMC promoter, expression of both SSTR2A and FUSl are linked. Thus, HA-SSTR2 or related genes may be expressed with a gene of interest such as a therapeutic gene. For example, the therapeutic gene can be an anti-cancer gene, such as FUSl. The construct set forth in FIG. 15 includes the hTERT promoter, a mini- cytomegalovirus (miniCMV) promoter, FUSl gene, an internal ribosome entry site, and a gene chimera consisting of a hemmaglutinin A tag and a somatostatin receptor type 2A (HA- SSTR2A) for amplified hTERT mediated expression of HA-SSTR2, a reporter, and of FUSl, a gene of interest. Other constructs have been created, including the hTERT promoter, a miniCMV promoter, a gene chimera consisting of a hemmaglutinin A tag and a somatostatin receptor type 2A (HA-SSTR2A) for amplified hTERT mediated expression of HA-SSTR2A only; and, for example, a mini-cytomegalovirus (miniCMV) promoter, a gene chimera consisting of a hemmaglutinin A tag and a somatostatin receptor type 2A with a C-terminus deletion for amplified hTERT mediated expression of HA-SSTR2 with the C-terminus deletion only.

Studies were also conducted to evaluate whether human miniCMV-hTERT promoter increases tissue specific expression by the hTERT promoter in mice bearing intrathoracic tumors. Mice bearing intrathoracic tumors derived from N417 cells were injected intravenously with plasmid-lipoplexes (20 micrograms DNA: 40 nmol DOTAP : Cholesterol/mouse). Plasmids containing a CMV promoter-enhanced green fluorescent protein (EGFP) resulted in expression in tumor and normal tissues (FIG. 16). Plasmids containing an hTERT based promoter resulted in expression in tumors, but not or minimally in normal tissue (FIG. 16). The hTMC promoter resulted in greater tissue specific expression than did the unamplified hTERT promoter (FIG. 16). Frozen sections of the various tissues were fixed in 4% formaldehyde and expression of EGFP was examined using a fluorescence microscope. Representative images are displayed in FIG. 16.

As set forth above, driven by a hTMC or a CMV promoter, expression of both SSTR2A and FUSl may be linked. Thus, HA-S STR2 or related genes may be expressed with a gene of interest such as the therapeutic gene FUSl. Lung cancer cells H 1299 transfected with plasmid containing a CMV promoter HA-SSTR2 insert results in expression of the HA- SSTR2 fusion protein (FIG. 17). H 1299 cells transfected with plasmid containing a hTMC promoter FUS1-HA-SSTR2 insert results in expression of both HA-SSTR2 fusion protein and FUSl (FIG. 17). H 1299 cells were transiently transfected and evaluated for fluorescence 72 hours later. The HA-S STR2 fusion protein was visualized via an antibody targeting the HA- domain and expression was seen as expected at the cell membrane. FUS 1 was visualized via an antibody to FUSl. The overlay demonstrated colocalization of the nuclear staining and HA-SSTR2 (CMV-HA-SSTR2) or colocalization of nuclear staining, HA-SSTR2, and FUSl when FUSl and HA-SSTR2 are linked for expression via an internal ribosome entry site (IRES) (FIG. 17).

EXAMPLE 6 The hTERT Promoter Amplified by the GAL4-VP16 System Results in Tumor

Specific Expression in vivo

Injection of a nude mouse via the tail vein with adenovirus containing a CMV promoter-green fluorescent protein insert (negative control, FIG. 18A, left) was found to not result in expression of HA-SSTR2 in the liver. Normal washout of 111-In-octreotide was seen via the kidneys. A nude mouse injected with adenovirus containing a CMV promoter- HA-S STR2 insert (positive control, FIG. 18 A, middle), resulted in expression of HA-SSTR2 in the liver. A nude mouse injected with adenovirus containing a hTERT-Gal4-VP16 promoter-HA-SSTR2 insert (FIG. 18A, right) did not result in expression in the liver, thus no expression is seen in normal tissue. A nude mouse bearing human fibrosarcoma tumors (derived from HTl 080 cells) was injected intratumorally with adenovirus containing a hTERT-Gal4-VP16 promoter-HA- SSTR2 insert in the left tumor or with adenovirus containing a CMV promoter-HA-SSTR2 insert in the right tumor. Expression was visualized in both tumors. Mice were injected with virus and two days later injected with 111 -In octreotide. Planar imaging was performed one day later. Representative images are displayed in FIG. 18B. These studies demonstrate that the hTERT promoter amplified by the Gal4-VP16 system results in tumor specific expression in vivo.

AU of the compositions and methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and methods, and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.

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Claims

1. A nucleic acid molecule comprising a coding region encoding a recombinant seven transmembrane G-protein associated receptor (GPCR) amino acid sequence, the coding region operatively linked to a tissue-selective promoter sequence.

2. The nucleic acid of claim 1, wherein the recombinant GPCR has a C-teminal deletion, has an altered internalization, is defective in intracellular signaling, or a combination thereof.

3. The nucleic acid of claim 1 or 2, wherein the GPCR is an acetylcholine receptor: Ml, M2, M3, M4, or M5; adenosine receptor: Al; A2A; A2B; or A3; adrenoceptors: alphalA, alphalB, alphalD, alpha2A, alpha2B, alpha2C betal, beta2, or beta3; angiotensin receptors: ATI, or AT2; bombesin receptors: BBl, BB2, or BB3; bradykinin receptors: Bl, B2, calcitonin, Ainilin, CGRP, or adrenomedullin receptors; cannabinoid receptors: CBl, or CB2; chemokine receptors: CCRl, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCRlO, CXCRl, CXCR2, CXCR3, CXCR4, CXCR5, CX3CR1, or XCRl; chemotactic receptors : C3a, C5a, or fMLP; cholecystokinin and gastrin receptors: CCKl, or CCK2; corticotropin-releasing factor receptors: CRFl, or CRF2; dopamine receptors: Dl, D2, D3, D4, or D5; endothelin receptors: ET(A) or ET(B); galanin receptors: GALl, GAL2, or GAL3; glutamate receptors: mgll, mgl2, mgl3, mgl4, mgl5, mgl6, mgl7, or mgl8; glycoprotein hormone receptors: FSH, LSH, or TSH; histamine receptors: Hl, H2, H3, or H4; 5-HT receptors: 5-HT1A, 5-HT1B, 5-HT1D, 5-HT1B, 5-HT1F, 5HT2A, 5-HT2F, 5-HT2C, 5- HT3, 5-HT4, 5-HT5A, 5-HT5B, 5-HT6, or 5-HT7; leukotriene receptors: BLT, CysLTl, or CysLT2; lysophospholipid receptors: edgl, edg2, edg3, or edg4; melanocorlin receptors: MCl; MC2; MC3; MC4, or MC5; melatonin receptors: MTl, MT2, or MT3; neuropeptide Y receptors: Yl, Y2, Y4, Y5, or Y6; neurotension receptors: NTSl, or NTS2; opioids: DOP, KOP, MOP, or NOP; P2Y receptors: P2Y1, P2Y2, P2Y4, P2Y6, P2Y11, or P2Y12); peroxisome proliferators: PPAR-alpha, PPAR-beta, or PPAR-gamma; prostanoid receptors: DP, FP, IP, TP, EPl, EP2, EP3, or EP4; protease-activated receptors: PARl, PAR2, PAR3, or PAR4; Somatostatin receptors: SSTRl, SSTR2, SSTR2A, SSTR3, SSTR4, or SSTR5; tachykinin receptors: NKl, NK2, or NK3; thyrotropin-releasing hormone receptors: TRHl, or TRH2; urotensin-II receptor; vasoactivate intestinal peptide or pituitary adenylate cyclase activating peptide receptors: VPACl, VPAC2, or PACl; or vasopressin or oxytocin receptors: Via, VIb, V2, or OT.

4. The nucleic acid of claim 3, wherein the GPCR is a somatostatin receptor.

5. The nucleic acid of claim 4, wherein the somatostatin receptor is a somatostatin receptor type 2A (SSTR2A).

6. The nucleic acid of claim 5, wherein the SSTR2A is signaling defective, has altered internalization, or a combination thereof.

7. The nucleic acid of claim 6, wherein the SSTR2A is truncated.

8. The nucleic acid of claim 7, wherein the SSTR2A is truncated carboxy terminal to amino acid 314.

9. The nucleic acid of claims 1-8, wherein the promoter sequence is a telomerase promoter, a human telomerase RNA (hTR) promoter, human telomerase reverse transcriptase promoter (hTERT) promoter, hTR operatively coupled to an amplification mechanism, or hTERT operatively coupled to an amplification mechanism.

10. The nucleic acid of claim 1-8, wherein the tissue-selective promoter or amplified tissue specific promoter is active in normal and/or diseased heart, lung, esophagus, muscle, intestine, breast, prostate, stomach, bladder, liver, spleen, pancreas, kidney, neurons, myocytes, leukocytes, immortalized cells, neoplastic cells, tumor cells, cancer cells, duodenum, jejunum, ileum, cecum, colon, rectum, salivary glands, gall bladder, urinary bladder, trachea, larynx, pharynx, aorta, arteries, capillaries, veins, thymus, lymph nodes, , bone marrow, pituitary gland, thyroid gland, parathyroid glands, adrenal glands, brain, cerebrum, cerebellum, medulla, pons, spinal cord, nerves, skeletal muscle, smooth muscle, bone, testes, epidiymides, prostate, seminal vesicles, penis, ovaries, uterus, mammary glands, vagina, skin, eyes, optic nerve, a promoter active in tissues derived from the same embryonic origin or one or more tissues effected by the same or similar disease.

11. The nucleic acid of claim 10, wherein the tissue selective promoter is active in a neoplastic cell, a tumor, or a cancer cell.

12. The nucleic acid of claim 11, wherein the cancer cell is a breast cancer cell, a lung cancer cell, a prostate cancer cell, an ovarian cancer cell, a brain cancer cell, a liver cancer cell, a cervical cancer cell, a colon cancer cell, a renal cancer cell, a skin cancer cell, a head and neck cancer cell, a bone cancer cell, an esophageal cancer cell, a bladder cancer cell, a uterine cancer cell, a lymphatic cancer cell, a stomach cancer cell, a pancreatic cancer cell, a testicular cancer cell, a lymphoma cell, or a leukemic cell.

13. The nucleic acid of claim 11, wherein the promoter sequence is an hTR promoter sequence, hTERT promoter sequence, CEA promoter sequence, a PSA promoter sequence, a probasin promoter sequence, a ARR2PB promoter sequence, AFP promoter sequence, MUC-I, MUC-4, mucin-like glycoprotein, C-erbB2/neu oncogene, Cyclo- oxygenase, E2F transcription factor 1, tyrosinase related protein, tyrosinase, or survivin, Tcfl- alpha, Ras, Raf, cyclin E, Cdc25A, HK II, KRT19, TFFl, SELlL, or an CEL.

14. The nucleic acid of claim 13, wherein the promoter sequence is an hTERT promoter sequence that is functional in a cancer cell.

15. The nucleic acid of claim 10, wherein the promoter sequence is an immunoglobulin heavy chain promoter sequence, an immunoglobulin light chain promoter sequence, a T-cell receptor promoter sequence, an HLA DQ a promoter sequence, an HLA DQ beta promoter sequence, a beta-interferon promoter sequence, an interleukin-2 promoter sequence, an interleukin-2 receptor promoter sequence, an MHC Class II 5 promoter sequence, an MHC Class II HLA-Dra promoter sequence, a beta-actin promoter sequence, a muscle creatine kinase (MCK) promoter sequence, a prealbumin (transthyretin) promoter sequence, an elastase I promoter sequence, a metallothionein (MTII) promoter sequence, a collagenase promoter sequence, an albumin promoter sequence, an alpha-fetoprotein promoter sequence, a gamma-globin promoter sequence, a beta-globin promoter sequence, a c-fos promoter sequence, a c-HA-ras promoter sequence, an insulin promoter sequence, a neural cell adhesion molecule (NCAM) promoter sequence, an alpha-1 -antitrypsin promoter sequence, an H2B (TH2B) histone promoter sequence, a type I collagen promoter sequence, a GRP94 promoter sequence, a GRP78 promoter sequence, an other glucose-regulated protein promoter sequence, a growth hormone promoter sequence, a human serum amyoid A (SAA) promoter sequence, a troponin I (TN I) promoter sequence, a platelet-derived growth factor (PDGF) promoter sequence, a Duchenne Muscular Dystrophy promoter sequence, an SV40 promoter sequence, a polyoma promoter sequence, a retrovirus promoter sequence, a papilloma virus promoter sequence, a Hepatitis B virus promoter sequence, a Human Immunodeficiency Virus promoter sequence, a Cytomegalovirus promoter sequence, a Gibbon Ape Leukemia Virus promoter sequence, a human LIMK2 gene promoter sequence, a somatostatin receptor promoter sequence, a murine epididymal retinoic acid-binding gene promoter sequence, a human CD4 promoter sequence, a mouse alpha2 (XI) collagen promoter sequence, a DlA dopamine receptor promoter sequence, an insulin-like growth factor II promoter sequence, human platelet endothelial cell adhesion molecule- 1 promoter sequence, a human alpha-lactalbumin promoter sequence, a 7SL promoter sequence, a human Y promoter sequence, a human MRP-7-2 promoter sequence, a 5S ribosomal promoter sequence, alpha fetoprotein, monocyt receptor for bacterial LPS, leukosialin, Sialophorin, leukocyte common antigen, Macrosialin or human analogue of macrosialin, Desmin, Elastase, Elastase I, Endoglin, fibronectin, VEGF receptors, glial fibrillary acidic protein, intercellular adhesion molecule 2, interferon beta, myoglobin, osteocalcin 2, prostate specific antigen, prostate specific membrane antigen, surfactant protein B, Synapsin, tyrosinase related protein, tyrosinase, or a functional hybrid, functional portion, or a combination of any of tissue/disease /lineage specific promoter sequences.

16. The nucleic acid of claim 1 - 8, wherein the tissue-selective promoter sequence is operatively coupled to a core promoter sequence.

17. The nucleic acid of claim 16, wherein the core promoter sequence is derived from a constitutive promoter such as ubiquitin promoter, an actin promoter, an elongation factor 1 alpha, an early growth factor response 1, an eukaryotic initiation factor 4Al, a ferritin heavy chain, a ferritin light chain, a glyceraldehyde 3 -phosphate dehydrogenase, a glucose- regulated protein 78, a glucose-regulated protein 94, a heat shock protein 70, a heat shock protein 90, a beta-kinesin, a phosphoglycerate kinase, an ubiquitin B, a beta-actin or a minimal viral promoter sequence.

18. The nucleic acid of claim 17, wherein the minimal viral promoter sequence is a RNA virus promoter, DNA virus promoter, adenoviral promoter sequence, a baculoviral promoter sequence, a CMV promoter sequence, a parvovirus promoter sequence, a herpesvirus promoter sequence, a poxvirus promoter sequence, an adeno-associated virus promoter sequence, a semiliki forest virus promoter sequence, an SV40 promoter sequence, a vaccinia virus promoter sequence, a lentivirus promoter, or a retrovirus promoter sequence

19. The nucleic acid of claim 18, wherein the minimal viral promoter sequence is a mini-CMV promoter sequence.

20. The nucleic acid of claim 19, wherein the tissue selective promoter is the hTERT promoter.

21. The nucleic acid of claim 20, wherein the hTERT promoter is operatively coupled to a first reporter.

22. The nucleic acid of claim 21, further comprising a second coding sequence wherein the second coding sequence encodes a therapeutic, a selectable marker, a recombinant transactivator, or a second imaging gene (reporter).

23. The nucleic acid of claim 22, comprising a first reporter is SSTR2 and a second reporter that is green fluorescent protein (GFP).

24. The nucleic acid of claim 1 - 8, further comprising a second coding sequence operatively coupled to a second tissue-selective promoter sequence, or an amplified tissue specific promoter or a non-selective promoter such as a CMV promoter.

25. The nucleic acid of claim 22, wherein the second coding sequence encodes a therapeutic, a selectable marker, a recombinant transactivator, or a second imaging gene (reporter).

26. The nucleic acid of claim 25, wherein the therapeutic is a tumor suppressor, an inducer apoptosis, an enzyme, a structural protein, a receptor, an antibody, an antibody fragment, a siRNA, a hormone, a paracrine factor, or an immunostimulant.

27. The nucleic acid of claim 26, wherein the tumor suppressor is FUS 1.

28. The nucleic acid of claim 25, wherein the selectable marker is a drug selection marker, an enzyme, a structural protein, a receptor, a paracrine factor, an immunologic marker, or a fluorescent protein.

29. The nucleic acid of claim 1, further comprising a second coding sequence, wherein the second coding sequence and the nucleic acid encoding the reporter are operatively linked.

30. The nucleic acid of claim 1, further comprising a second coding sequence, wherein the second coding sequence and the nucleic acid encoding the recombinant GPCR amino acid sequence are operatively coupled to a bidirectional promoter.

31. The nucleic acid of claim 1, further comprising a second coding sequence, wherein the second coding sequence and the nucleic acid encoding the recombinant seven transmembrane G-protein associated receptor amino acid sequence are separated by an IRES.

32. The nucleic acid of claim 29-31, wherein the second coding sequence encodes a therapeutic, a selectable marker, or a reporter.

33. The nucleic acid of claim 32, wherein the therapeutic is a tumor suppressor, an inducer apoptosis, an enzyme, an antibody, a hormone, or an immunostimulant.

34. The nucleic acid of claim 33 , wherein the tumor suppressor is FUS 1.

35. The nucleic acid of claim 32, wherein the selectable marker is a drug selection marker, an enzyme, an immunologic marker, or a fluorescent protein.

36. The nucleic acid of claim 1 - 8, further comprising a protein tag fused to the N- terminal end or C-terminal end of the GPCR amino acid sequence.

37. The nucleic acid of claim 36, wherein said protein tag has enzymatic activity.

38. The nucleic acid of claim 37, wherein the protein tag is hemagglutinin A, beta- galactosidase, thymidine kinase, transferrin, myc-tag, VP 16, (His)₆-tag, FLAG, or chloramphenicol acetyl transferase.

39. A nucleic acid comprising a nucleic acid sequence encoding a reporter that is detectable in a subject by non-invasive methods operatively coupled to a promoter sequence that binds a recombinant transactivator.

40. The nucleic acid of claim 39, wherein the recombinant transactivator is Gal4VP16.

41. The nucleic acid of claim 39, further comprising a second coding sequence.

42. The nucleic acid of claim 39, wherein the reporter encoding sequence and the second coding sequence are separated by an IRES.

43. The nucleic acid of claim 41, wherein the second coding sequence encodes a therapeutic, a selectable marker, or a reporter.

44. The nucleic acid sequence of claim 41, further comprising a third coding sequence operatively coupled to a tissue-selective promoter, wherein the third coding sequence encodes a recombinant transactivator.

45. The nucleic acid of claim 41, wherein the second coding sequence is operatively coupled to a tissue-selective promoter, wherein the second coding sequence encodes a recombinant transactivator.

46. The nucleic acid of claim 39, comprised in a delivery vehicle.

47. The nucleic acid of claim 46, wherein the delivery vehicle is a lipid, a liposome, a plasmid, a viral vector, a phage, a polyamino acid such as polylysine, a prokaryotic cell, or a eukaryotic cell.

48. A method of imaging or treating cells in a subject comprising: a) introducing a nucleic acid encoding a reporter detectable in vivo using non-invasive methods operatively coupled to a tissue-selective promoter or an amplified tissue specific promoter to a subject; and b) subjecting the subject to a non-invasive imaging technique or a therapeutic that selectively interacts with the reporter.

49. The method of claim 48, wherein the therapeutic is a radiotherapeutic, a siRNA, a prodrug, or a chemotherapeutic.

50. The method of claim 48, wherein the tissue-selective promoter is active in normal or diseased heart, lung, esophagus, muscle, intestine, breast, prostate, stomach, bladder, liver, spleen, pancreas, kidney, neurons, myocytes, leukocytes, immortalized cells, neoplastic cells, tumor cells, cancer cells, duodenum, jejunum, ileum, cecum, colon, rectum, salivary glands, gall bladder, urinary bladder, trachea, larynx, pharynx, aorta, arteries, capillaries, veins, thymus, lymph nodes, , bone marrow, pituitary gland, thyroid gland, parathyroid glands, adrenal glands, brain, cerebrum, cerebellum, medulla, pons, spinal cord, nerves, skeletal muscle, smooth muscle, bone, testes, epidiymides, prostate, seminal vesicles, penis, ovaries, uterus, mammary glands, vagina, skin, eyes, optic nerve, tissues derived from the same embryonic origin or tissues effected by the same or similar disease.

51. The method of claim 50, wherein the tissue selective promoter is active in a neoplastic cell, a tumor cell, or a cancer cell.

52. The method of claim 51, wherein the cancer cell is a breast cancer cell, a lung cancer cell, a prostate cancer cell, an ovarian cancer cell, a brain cancer cell, a liver cancer cell, a cervical cancer cell, a colon cancer cell, a renal cancer cell, a skin cancer cell, a head and neck cancer cell, a bone cancer cell, an esophageal cancer cell, a bladder cancer cell, a uterine cancer cell, a lymphatic cancer cell, a stomach cancer cell, a pancreatic cancer cell, a testicular cancer cell, a lymphoma cell, or a leukemic cell.

53. The method of claim 51, wherein the promoter sequence is an hTR, hTERT promoter sequence, CEA promoter sequence, a PSA promoter sequence, a probasin promoter sequence, a ARR2PB promoter sequence, AFP promoter sequence, MUC-I, MUC-4, mucin- like glycoprotein, C-erbB2/neu oncogene, Cyclo-oxygenase, E2F transcription factor 1, tyrosinase related protein, tyrosinase, or survivin, Tcfl -alpha, Ras, Raf, cyclin E, Cdc25A, HK II, KRTl 9, TFFl, SELlL, or an CEL.

54. The method of claim 53, wherein the promoter sequence is an hTERT promoter sequence.

55. The method of claim 50, wherein the promoter sequence is an immunoglobulin heavy chain promoter sequence, an immunoglobulin light chain promoter sequence, a T-cell receptor promoter sequence, an HLA DQ a promoter sequence, an HLA DQ beta promoter sequence, a beta-interferon promoter sequence, an interleukin-2 promoter sequence, an interleukin-2 receptor promoter sequence, an MHC Class II 5 promoter sequence, an MHC Class II HLA-Dra promoter sequence, a beta-actin promoter sequence, a muscle creatine kinase (MCK) promoter sequence, a prealbumin (transthyretin) promoter sequence, an elastase I promoter sequence, a metallothionein (MTII) promoter sequence, a collagenase promoter sequence, an albumin promoter sequence, an alpha-fetoprotein promoter sequence, a gamma-globin promoter sequence, a beta-globin promoter sequence, a c-fos promoter sequence, a c-HA-ras promoter sequence, an insulin promoter sequence, a neural cell adhesion molecule (NCAM) promoter sequence, an alpha- 1 -antitrypsin promoter sequence, an H2B (TH2B) histone promoter sequence, a type I collagen promoter sequence, a GRP94 promoter sequence, a GRP78 promoter sequence, an other glucose-regulated protein promoter sequence, a growth hormone promoter sequence, a human serum amyoid A (SAA) promoter sequence, a troponin I (TN I) promoter sequence, a platelet-derived growth factor (PDGF) promoter sequence, a Duchenne Muscular Dystrophy promoter sequence, an SV40 promoter sequence, a polyoma promoter sequence, a retrovirus promoter sequence, a papilloma virus promoter sequence, a Hepatitis B virus promoter sequence, a Human Immunodeficiency Virus promoter sequence, a Cytomegalovirus promoter sequence, a Gibbon Ape Leukemia Virus promoter sequence, a human LIMK2 gene promoter sequence, a somatostatin receptor promoter sequence, a murine epididymal retinoic acid-binding gene promoter sequence, a human CD4 promoter sequence, a mouse alpha2 (XI) collagen promoter sequence, a DlA dopamine receptor promoter sequence, an insulin-like growth factor II promoter sequence, human platelet endothelial cell adhesion molecule- 1 promoter sequence, a human alpha-lactalbumin promoter sequence, a 7SL promoter sequence, a human Y promoter sequence, a human MRP-7-2 promoter sequence, a 5 S ribosomal promoter sequence, alpha fetoprotein, monocyt receptor for bacterial LPS, leukosialin, Sialophorin, leukocyte common antigen, Macrosialin or human analogue of macrosialin, Desmin, Elastase, Elastase I, Endoglin, fibronectin, VEGF receptors, glial fibrillary acidic protein, intercellular adhesion molecule 2, interferon beta, myoglobin, osteocalcin 2, prostate specific antigen, prostate specific membrane antigen, surfactant protein B, Synapsin, tyrosinase related protein, tyrosinase, or a functional hybrid, functional portion, or a combination of any of tissue, disease , or lineage specific promoter sequences.

56. The method of claim 48, wherein non-invasive imaging comprises detecting expression of the reporter by assaying for an association between the cells and a detectable moiety.

57. The method of claim 56, wherein the association between the cells expressing the reporter and a detectable moiety comprises binding of the detectable moiety by the cells, binding of a ligand operably coupled to the detectable moiety by the cells, cellular uptake of the detectable moiety, or cellular uptake of a ligand operably coupled to the detectable moiety.

58. The method of claim 56, wherein the detectable moiety is a protein, a radioisotope, a fluorophore, a visible light emitting fiuorophore, near infrared light emitting fluorophore, infrared light emitting fluorophore, a metal, a ferromagnetic substance, a substance with a specific MR spectroscopic signature, an X-ray absorbing or reflecting substance, a sound altering substance, or an electromagnetic emitting substance.

59. The method of claim 56, wherein the detectable moiety is operably coupled to a ligand that specifically binds the reporter.

60. The method of claim 59, wherein the ligand is a nucleic acid such as a DNA or RNA molecule, a protein, a polypeptide, a peptide, an antibody, an antibody fragment, or a small molecule.

61. The method of claim 48, wherein non-invasive methods comprise MRI, MR spectroscopy, radiography, CT, ultrasound, planar gamma camera imaging, SPECT, PET, other nuclear medicine-based imaging, optical imaging using visible light, optical imaging using luciferase, optical imaging using a fluorophore, other optical imaging, imaging using near infrared light, or imaging using infrared light.

62. The method of claim 48, further comprising imaging a tissue during a surgical procedure on the subject.

63. The method of claim 48, wherein the subject is a cancer patient.

64. The method of claim 63, wherein the nucleic acid encodes a therapeutic for treating a cancer patient.

65. The method of claim 62, wherein cancer patient is undergoing a second anticancer therapy.