WO2006099019A2 - Methodes et composition relatives a l'imagerie in vivo d'une expression genique - Google Patents

Methodes et composition relatives a l'imagerie in vivo d'une expression genique Download PDF

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WO2006099019A2
WO2006099019A2 PCT/US2006/008374 US2006008374W WO2006099019A2 WO 2006099019 A2 WO2006099019 A2 WO 2006099019A2 US 2006008374 W US2006008374 W US 2006008374W WO 2006099019 A2 WO2006099019 A2 WO 2006099019A2
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Prior art keywords
promoter sequence
promoter
nucleic acid
sequence
cell
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PCT/US2006/008374
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WO2006099019A3 (fr
WO2006099019A8 (fr
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Vikas Kundra
Bingliang Fang
Lin X. Ji
Dan Yang
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Board Of Regents, The University Of Texas System
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Priority to EP06748324A priority Critical patent/EP1866421A2/fr
Priority to CA002601243A priority patent/CA2601243A1/fr
Priority to AU2006223435A priority patent/AU2006223435A1/en
Priority to JP2008500900A priority patent/JP2008532519A/ja
Publication of WO2006099019A2 publication Critical patent/WO2006099019A2/fr
Publication of WO2006099019A3 publication Critical patent/WO2006099019A3/fr
Publication of WO2006099019A8 publication Critical patent/WO2006099019A8/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/0002—General or multifunctional contrast agents, e.g. chelated agents
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/008—Vector systems having a special element relevant for transcription cell type or tissue specific enhancer/promoter combination
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/15—Vector systems having a special element relevant for transcription chimeric enhancer/promoter combination
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/60—Vector systems having a special element relevant for transcription from viruses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/80—Vector systems having a special element relevant for transcription from vertebrates
    • C12N2830/85—Vector systems having a special element relevant for transcription from vertebrates mammalian
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2840/00—Vectors comprising a special translation-regulating system
    • C12N2840/20—Vectors comprising a special translation-regulating system translation of more than one cistron
    • C12N2840/203—Vectors comprising a special translation-regulating system translation of more than one cistron having an IRES

Definitions

  • 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.
  • GPCR seven transmembrane G- protein associated receptor
  • 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.
  • imaging may not be targeted to a tissue of interest, such as a tumor.
  • imaging modality such as anatomical and functional imaging technology
  • imaging modality such as anatomical and functional imaging technology
  • 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.
  • 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.
  • a tumor-specific promoter-driven therapeutic nucleic acid may be coupled to a reporter, for example, via a bifunctional reporter or via an IRES.
  • 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.
  • 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.
  • the gene therapy is administered systemically.
  • 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.
  • An aspect of the invention includes expression amplification strategies for increasing expression of a reporter and/or a therapeutic in a cell of interest.
  • 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.
  • the tissue selective promoter is an hTERT promoter, and the hTERT promoter is operatively coupled to a first reporter.
  • the hTERT promoter sequence may be SEQ ID NO:1.
  • 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.
  • 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.
  • GFP green fluorescent protein
  • 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.
  • 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 64 Cu, 68 Ga, 18 F, 111 In or 99m Tc.
  • 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.
  • GPCR seven transmembrane G-protein associated receptor
  • 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.
  • 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
  • the GPCR is a somatostatin receptor, preferably a somatostatin receptor type 2A (SSTR2A).
  • SSTR2A somatostatin receptor type 2A
  • the SSTR2A has a C- terminus deletion and/or has altered internalization and/or is signaling defective.
  • 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.
  • 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.
  • 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,
  • 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.
  • 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.
  • 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,
  • 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 ⁇ promoter sequence, an HLA DQ ⁇ 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-f
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the minimal viral promoter sequence is a mini-CMV promoter sequence.
  • the mini-CMV promoter sequence can be SEQ ID NO:2.
  • the tissue selective promoter is the hTERT promoter.
  • 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.
  • 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.
  • 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.
  • 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, pl ⁇ , p21, p57, p27, and BRCA2.
  • 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.
  • the selectable marker may be a drug selection marker, an enzyme, an immunologic marker, or a fluorescent protein.
  • 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.
  • 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.
  • 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.
  • 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-X 3 , Bik, Bid, Harakiri, Ad ElB, Bad, and ICE-CED2 protease.
  • the tumor suppressor is FUSl.
  • 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.
  • 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.
  • tk thymidine kinase
  • CAD CAD- deficient cells
  • hprt mammalian hypoxanthine-guanine phosphoribosyl transferase
  • 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.
  • GFP green fluorescent protein
  • EGFP enhanced green fluorescent protein
  • GFPmut2 Renilla Reniformis green fluorescent protein
  • EYFP enhanced yellow fluorescent protein
  • EYFP enhanced cyan fluorescent protein
  • EBFP enhanced blue fluorescent protein
  • dsRED citrine and red fluorescent protein from discosoma
  • 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.
  • 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.
  • 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.
  • histidine residues e.g., 4 to 8 consecutive histidine residues
  • 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.
  • FIG. 1 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.
  • the recombinant transactivator is Gal4VP16.
  • the nucleic acid may or may not comprise a second coding sequence.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • a detectable moiety is any molecule or agent that can emit a signal that is detectable by non-invasive imaging.
  • 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.
  • the detectable moiety is operably coupled to a ligand that specifically binds the reporter.
  • 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.
  • 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.
  • 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.
  • the subject is a cancer patient.
  • the nucleic acid may or may not encode a therapeutic for treating a cancer patient.
  • the cancer patient is undergoing an anticancer therapy.
  • anticancer therapies include chemotherapy, radiation therapy, surgical therapy, immunotherapy, and gene therapy.
  • a or “an” may mean one or more.
  • the words “a” or “an” when used in conjunction with the word “comprising”, the words “a” or “an” may mean one or more than one.
  • another may mean at least a second or more.
  • 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 ⁇ .05.
  • FIG. IB Western blot. Twenty micrograms of protein were loaded per lane.
  • FIG. 1C Receptor binding assay.
  • FIGs. 3A-3G ⁇ -camera imaging.
  • 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 111 Li-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.
  • G The size of the object on the planar image may not correlate with the true anatomic size of the object.
  • 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. 4C A representative T2 FSE image of a subcutaneous tumor in the left shoulder.
  • 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.
  • 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
  • FIG. 8A Lung N417 tumors were established by intrathoracic injection of 10 6 N417 cells in nude mice. Mice were treated with various EGFP-nanoparticles (20 ⁇ g
  • DNA 40 nmmol DOTAP: Choi/mouse
  • 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. 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).
  • HTl 080 human fibrosarcoma
  • 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.
  • hTERT human telomerase promoter
  • miniCMV mini cytomegalovirus
  • FUSl gene an internal ribosome entry site
  • 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
  • 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
  • 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.
  • 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).
  • CMV-HA-S STR2 colocalization of the nuclear staining and HA-S STR2
  • FUSl internal ribosome entry site
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the gene therapy is administered systemically.
  • 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.
  • 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 in particular may serve to diagnose and stage cancer.
  • 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).
  • 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).
  • PET nuclear imaging
  • PET gamma camera
  • in vivo optical imaging systems Reviewed in Blasberg et al, 2003.
  • 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).
  • 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).
  • 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.
  • 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.
  • imaging is performed concurrently with administration of a therapeutic gene to a subject.
  • 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.
  • 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.
  • 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.
  • 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.
  • the ligand is a radiolabeled probe, such as ⁇ ⁇ -octreotide.
  • 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.
  • measuring a signal can involve use of gamma-camera imaging of an 111 In or 99m Tc conjugate, in particular 111 In- octreotide or 99m Tc-somatostatin analogue.
  • CT Computerized tomography
  • 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.
  • 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.
  • contrast agents aid in assessing the vascularity of a soft tissue or bone lesion.
  • 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.
  • 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).
  • gadopentate agents has been used as a CT contrast agent
  • Magnetic resonance imaging 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.
  • MRI Magnetic resonance imaging
  • 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.
  • RF radio frequency
  • Various magnetic field gradients and other RF pulses then act on the spins to code spatial information into the recorded signals.
  • 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.
  • CT and MRI provide anatomical information that aid in distinguishing tissue boundaries and vascular structure.
  • 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).
  • 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).
  • PET positron emission tomography
  • SPECT single- photon emission computed tomography
  • 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.
  • SPECT single-photon emission computed tomography
  • 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 11 C, 13 N, 15 O, 18 F, 82 Rb, 62 Cu, and 68 Ga.
  • SPECT radiopharmaceuticals are commonly labeled with positron emitters such as 99m Tc, 201 Tl, and 67 Ga, 111 In.
  • 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 99m TcO4- DTPA, 201 Tl, and [ 67 Ga]citrate are excluded by normal brain cells, but enter into tumor cells because of altered BBB.
  • SPECT perfusion agents such as [ 123 I]IMP, [ 99m Tc]HMP AO, [ 99m Tc]ECD are lipophilic agents, and therefore diffuse into the normal brain.
  • Important receptor-binding SPECT radiopharmaceuticals include [ 123 I]QNE, [ 123 I]IBZM, and [ 123 I]iomazenil. These tracers bind to specific receptors, and are of importance in the evaluation of receptor-related diseases
  • 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.
  • 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.
  • 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 include perfluorine and perfluorine analogs, which are designed to overcome these limitations by helping to enhance grey-scale images and Doppler signals.
  • the imaging modality may include, but are not limited to, CT, MRI, PET, SPECT, ultrasound, or optical imaging.
  • CT computed tomography
  • MRI magnetic resonance imaging
  • PET magnetic resonance imaging
  • SPECT positron emission tomography
  • ultrasound magnetic resonance imaging
  • 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.
  • the imaging studies may be performed during administration of the dual imaging compound of the present invention, or at any time thereafter.
  • 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.
  • a second imaging modality may be performed concurrently with the first imaging modality, or at any time following the first imaging modality.
  • 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.
  • reporter genes either alone or in conjunction with a gene of interest.
  • the therapeutic gene may be fused to the reporter or be produced as a separate protein.
  • 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 .
  • 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.
  • the reporter may be used to follow cell trafficking.
  • specific cells may be transfected with a reporter and then returned to an animal to assess homing.
  • Costa et al. (2001) transferred myelin basic protein-specific CD4+ T cells that were transduced to express IL- 12 p40 and luciferase.
  • luciferase was used to demonstrate trafficking to the central nervous system.
  • IL- 12 p40 inhibited inflammation.
  • PET positron emission tomography
  • EBV Epstein-Barr virus
  • HSV-TK herpes simplex virus- 1 thymidine kinase
  • 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.
  • 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.
  • HSV-TK 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.
  • 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.
  • 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.
  • adeno-associated virus delivery systems support inserts of approximately 3 kb.
  • 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.
  • 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.
  • 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.
  • inhibition of cell growth would not be desirable in cell based therapies where cell implantation and growth is desired.
  • somatostatin receptor based reporters such as delta 314 are needed that are deficient in signaling but that can still be imaged in vivo.
  • reporter refers to any genetic sequence or encoded polypeptide sequence that is detectable and distinguishable from other genetic sequences or encoded polypeptides present in cells.
  • 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.
  • 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.
  • 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.
  • 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 ⁇ -lactamase, ⁇ -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.
  • GPCRs G- protein coupled receptors
  • somatostatin receptors CD2, CD4, CD8
  • symporters such as NIS
  • aspects of the invention include creating signaling deficient reporters by creating and comparing receptor mutants.
  • 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.
  • a ligand such as octreotide
  • 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.
  • HA hemagglutinin-A
  • the tag 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.
  • the reporter amino acid sequence is a GPCR. GPCRs are discussed at length elsewhere in this specification.
  • 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.
  • 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).
  • GFP green fluorescent protein
  • EGFP enhanced green fluorescent protein
  • 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).
  • a reporter nucleic acid may encode a polypeptide having a tag.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • GPCRs Recombinant G-Protein Coupled Receptors
  • GPCRs transmembrane G-protein associated receptors
  • SPCRs transmembrane G-protein associated receptors
  • 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,
  • the GPCR is a somatostatin receptor, such as a somatostatin type 2 receptor.
  • somatostatin receptors 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, extracellular 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 PCRTM.
  • the nucleic acid may be complementary DNA (cDNA).
  • cDNA is DNA prepared using messenger RNA (mRNA) as a template.
  • mRNA messenger RNA
  • a cDNA does not contain any interrupted coding sequences and usually contains almost exclusively the coding region(s) for the corresponding protein.
  • the nucleic acid may be produced synthetically.
  • genomic DNA may be combined with cDNA or synthetic sequences to generate specific constructs.
  • 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,
  • 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.
  • 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
  • SSTR somatostatin receptors
  • 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, 111 In 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.
  • 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.
  • 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.
  • 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.
  • SSTR2 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.
  • the role of the MAP kinase pathway in mediating inhibition of proliferation by SSTR2 is not yet clear.
  • nNOS neuronal nitric oxide synthase
  • 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-PTP ⁇ 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 .
  • the C-terminus and intracytoplasmic domains of SSTR2 appear to be involved.
  • deletion analysis has demonstrated that the cytoplasmic C-terminus regulates inhibition of cAMP production.
  • deletion of the SSTR2 after amino acid 314 is signaling defective and can be imaged in vivo.
  • 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.
  • 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.
  • a ligand may be contacted with the cell for imaging.
  • the ligand may or may not be internalized by the cell.
  • the ligand may bind to or associate with the reporter. Any method of binding of the ligand to the reporter is contemplated by the present invention.
  • 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.
  • the ligand is capable of binding to or being coupled to a molecule or part of a molecule that can be imaged.
  • 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.
  • the ligand may be 111 In- octreotide.
  • 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.
  • the ligand may be capable of binding to or being coupled to a CT contrast agent or an MRI contrast agent.
  • 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.
  • the ligand can bind to a protein tag that is fused to the reporter.
  • 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 67 Ga, 68 Ga, "'"Tc, 111 In, 123 I, 125 1, 131 I, 169 Yb, 60 Cu, 61 Cu, 64 Cu, 62 Cu, 201 Tl, 72 A, and 157 Gd. Due to better imaging characteristics and lower price, attempts have been made to replace the 123 I, 131 I, 67 Ga and 111 In labeled compounds with corresponding 99m Tc labeled compounds when possible. Due to favorable physical characteristics as well as low price, PPm Tc has been preferred to label radiopharmaceuticals .
  • 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.
  • 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.
  • m Tc 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.
  • a molybdenum- 99/technetium-PPm generator One of ordinary skill in the art would be familiar with methods to determine optimal radioimaging in humans.
  • 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.
  • 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.
  • 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 99m Tc 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.
  • the radiolabel is administered by any method known to those of ordinary skill in the art.
  • administration may be in a single unit injectable dose, administered as a radiolabeled ligand.
  • 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.
  • 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.
  • imaging may be performed using any method known to those of ordinary skill in the art. Examples include PET, SPECT, and 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).
  • 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.
  • 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.
  • 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.
  • 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).
  • the hTERT promoter may be used to specifically localize gene expression in tumors.
  • 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.
  • 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.
  • 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
  • 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.
  • 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.
  • the therapeutic can be an anti-cancer agent.
  • the therapeutic is a radionuclide. Radionuclides are discussed in greater detail below.
  • 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.
  • 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.
  • 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.
  • 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).
  • nucleic acid encompass the terms “oligonucleotide” and “polynucleotide,” each as a subgenus of the term “nucleic acid.”
  • oligonucleotide refers to a molecule of between about 3 and about 100 nucleobases in length.
  • polynucleotide refers to at least one molecule of greater than about 100 nucleobases in length.
  • 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
  • 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.
  • 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.
  • promoter is used interchangeably with “promoter element” and “promoter sequence.”
  • 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.
  • a promoter sequence located proximally to the 5' end of a transgene coding sequence is usually operably linked with the transgene.
  • 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).
  • 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.
  • 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.
  • 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.”
  • an enhancer may be one naturally associated with a nucleic acid sequence, located either downstream or upstream of that sequence.
  • 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.
  • 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.
  • sequences may be produced using recombinant cloning and/or nucleic acid amplification technology, including PCRTM, in connection with the compositions disclosed herein (see U.S.
  • Such promoters may be used to drive reporter expression, which include, but are not limited to GPCRs, ⁇ -galactosidase or luciferase to name a few.
  • reporter expression include, but are not limited to GPCRs, ⁇ -galactosidase or luciferase to name a few.
  • 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.
  • 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.
  • tissue specific promoters including hTERT
  • 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.
  • a signaling deficient reporter is desirable in such a construct.
  • the tissue specific promoter is the telomerase promoter.
  • telomeres 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.
  • telomere 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • CEA carcinoembryonic antigen
  • hTERT 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.
  • CMV carcinoembryonic antigen
  • 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.
  • an imagable reporter such as, but not limited to those based on the somatostatin receptor type 2
  • Linking an imagable reporter such as, but not limited to those based on the somatostatin receptor type 2
  • 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.
  • 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.
  • 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.
  • telomere activity has 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.
  • 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.
  • a transactivator is a recombinant transactivator.
  • a recombinant transactivator may be expressed in cells into which a nucleic acid of the invention is introduced.
  • a recombinant transactivator or a nucleic acid encoding a recombinant transactivator may be introduced before, with or after a nucleic acid of the invention.
  • 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-
  • 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).
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 ⁇ and/or DQ ⁇ (Sullivan et al, 1987); ⁇ -Interferon (Goodbourn et al, 1986; Fujita
  • 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
  • IRES elements from two members of the picornavirus family Polio and encephalomyocarditis
  • IRES elements from a mammalian message Macejak and Sarnow, 1991
  • IRES elements can be linked to heterologous open reading frames.
  • each open reading frame can be transcribed together, each separated by an IRES, creating polycistronic messages.
  • IRES element 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.
  • 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.
  • 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
  • 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.
  • 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.
  • the termination signal may be a lack of transcribable or translatable sequence, such as due to a sequence truncation.
  • a vector in a host cell may contain one or more origins of replication sites (often termed "ori"), which is a specific nucleic acid sequence at which replication is initiated.
  • ori origins of replication sites
  • ARS autonomously replicating sequence
  • nucleic acid sequences that include one or more nucleic acid sequences of interest, also referred to as genes of interest.
  • 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.
  • 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.
  • 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.
  • a therapeutic gene can be a gene administered to a subject for treatment or prevention of diabetes or cancer.
  • 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 ⁇ , interferon ⁇ , interferon ⁇ , ADP, p53, ABLI, BLCl, B
  • 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.
  • tumor suppressor nucleic acids within this definition include, but are not limited to APC, CYLD, HIN-I, KRAS2b, pl ⁇ , 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.
  • 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 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.
  • 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.
  • 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.
  • cytokines 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- ⁇ and - ⁇ ; mullerian-inhibiting substance; mouse gonadotropin-associated peptide; inhibin; activin; vascular endothelial growth factor; integrin; thrombopoietin (TPO); nerve growth factors such as NGF- ⁇ ; platelet- growth factor; transforming growth factors (TGFs) such as TGF- ⁇ and TGF
  • 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,
  • 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, gly
  • 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, ⁇ -endorphin, ⁇ -melanocyte stimulating hormone, cholecystokinin, endothelin I, galanin, gastric inhibitory peptide, glucagon, insulin, lipotropins, neurophysins, somatostatin, calcitonin, calcitonin gene related peptide, ⁇ -calcitonin gene related peptide, hypercalcemia of malignancy factor, parathyroid hormone-related protein, parathyroid hormone-related protein, glucagon-like peptide, pancreastatin, pancreatic peptide, peptide YY,
  • 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.
  • therapeutic gene 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.
  • aspects of the invention include detecting nucleic acids delivered alone or in combination with a second imaging agent and/or a therapeutic agent.
  • the nucleic acid delivered includes a recombinant GPCR of the invention and in particular embodiments a SSTR.
  • compositions that include a delivery vehicle for delivery of the nucleic acid into a cell.
  • a delivery vehicle for delivery of the nucleic acid into a cell.
  • delivery vehicles for delivery of nucleic acids to a cell since these experimental methods are well-known in the art.
  • 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.
  • 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).
  • 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 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.
  • 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).
  • pIM45 McCarty et al, 1991; incorporated herein by reference
  • HSV Herpes simplex virus
  • 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.
  • the availability of different viral control sequences with varying performance 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.
  • 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.
  • viral vectors may be employed as constructs in the present invention.
  • 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.
  • Non-viral methods for the transfer of nucleic acids into cells 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).
  • 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.
  • a gene construct complexed with Lipofectamine (Gibco BRL).
  • 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).
  • lipid structures can be used to encapsulate compounds that are toxic (chemotherapeutics) or labile
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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":
  • 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.
  • CDDP cisplatin
  • carboplatin carbop
  • Radiotherapy Other factors that cause DNA damage and have been used extensively include what are commonly known as ⁇ -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.
  • immune modulators generally, rely on the use of immune modulators, immune effector cells and molecules to cure or palliate disease.
  • 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.
  • 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 could be used as part of a combined therapy, in conjunction with gene therapy.
  • the general approach for combined therapy is discussed below.
  • the tumor cell must bear some marker that is amenable to targeting, i.e., is not present on the majority of other cells.
  • 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.
  • 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.
  • 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.
  • compositions 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.
  • pharmaceutically 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.
  • 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.
  • compositions 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.
  • 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
  • compositions 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.
  • compositions may comprise, for example, at least about 0.1% of a dual imaging agent or a dual therapeutic agent.
  • 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.
  • 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
  • the composition may comprise various antioxidants to retard oxidation of one or more component.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • functional planar and SPECT imaging
  • MR anatomic
  • CA hemagglutinin A
  • HTl 080 cells human fibrosarcoma, ATCC, Rockville, MD
  • DMEM Dulbecco's Modified Eagle Medium
  • GPS penicillin, and streptomycin
  • FBS fetal bovine serum
  • 1 ⁇ g DNA was added with lipofectin 2000 (Invitrogen, Carlsbad, CA) to 1 x 10 5 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
  • 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.
  • 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.
  • 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.
  • mice underwent planar imaging for 10 minutes using a ⁇ -camera (mCAM, Siemens Medical Solutions, Hoffman Estates, IL) fitted with a medium-energy parallel hole collimator. No attenuation correction was used.
  • 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.
  • SPECT reconstruction Butterworth 0.6 Nyquist, 10 th order filtered-backprojection was performed.
  • 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 ⁇ l of different amounts of 1 ⁇ n-chloride (93-0.3 ⁇ Ci). 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) .
  • ROI region of interest
  • 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
  • receptor- binding assays were performed.
  • 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 111 In-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.
  • expression levels based on the anti-HA antibody corroborate receptor-binding data based on ⁇ 1 In-OCt.
  • FIG. 2 demonstrates ex vivo biodistribution analysis of 111 In-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 111 In-Od: is eliminated through the kidneys and liver and there was variability in the extent of each route used among the mice.
  • FIG. 3A 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.
  • 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.
  • 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 .
  • mice were also imaged by MR.
  • the pelvis image of a representative mouse shows that the tumor has intermediate signal and demonstrates excellent contrast with adjacent structures.
  • 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).
  • telomere reverse transcriptase telomerase reverse transcriptase
  • hTERT Human telomerase reverse transcriptase
  • 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).
  • hTERT-mini-CMV transcription-promoting strength of the hTERT promoter
  • hTMC has been engineered by optimally fusing essential hTERT promoter sequences with minimal CMV promoter elements.
  • 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.
  • FIG. 9A 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 111 In- 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).
  • FIG. 10 A novel tumor-specific dual reporter and therapeutic gene expression plasmid vector for molecular imaging has been developed (FIG. 10).
  • 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).
  • IRES Internal Ribosome Entry Site
  • hTERT promoter has been cloned and shown to be capable of targeting transgene expression in tumors but not in normal cells.
  • the weak transcription- promoting strength of hTERT promoter like most other intrinsic mammalian promoters, has hampered its direct use for cancer gene therapy.
  • hTMC novel chimera liTERT-mini-CMV
  • 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.
  • FI fluorescence imaging
  • hTMC-EGFP tumor-selectivity of EGFP expression driven by the hTERT promoter
  • 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.
  • N417 tumor mouse model was used to evaluate the therapeutic efficacy of systemic treatment with a novel hTMC-Fr ⁇ i-nanoparticle by non-invasive and quantitative MR imaging analysis.
  • a significant inhibition (PO.001) of tumor growth was detected in animals treated by hTMC-Fr ⁇ i-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.
  • 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.
  • HA-S STR2 A hemrnaglutinin A tag and somatostatin receptor type 2 A
  • 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.
  • 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.
  • 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.
  • FIG. 15 demonstrated schematically a plasmid map showing that driven by an amplified hTMC promoter, expression of both SSTR2A and FUSl are linked.
  • HA-SSTR2 or related genes may be expressed with a gene of interest such as a therapeutic gene.
  • 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.
  • miniCMV mini- cytomegalovirus
  • FUSl gene an internal ribosome entry site
  • 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.
  • 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.
  • miniCMV mini-cytomegalovirus
  • mice bearing intrathoracic tumors 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).
  • EGFP CMV promoter-enhanced green fluorescent protein
  • 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.
  • 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).
  • 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.
  • 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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Abstract

L'invention concerne des séquences d'acides nucléiques codant pour une séquence d'acide aminé du récepteur de recombinaison à sept transmembranes couplé à une protéine G (GPCR), fonctionnellement couplée à une séquence de promoteur sélectif d'un tissu. L'invention concerne également des méthodes d'imagerie ou de traitement de cellules chez un sujet impliquant l'introduction d'un acide nucléique codant pour un reporter détectable in vivo à l'aide de méthodes non invasives couplé à un promoteur sélectif d'un tissu ou à un promoteur sélectif d'un tissu amplifié du sujet, et la soumission dudit sujet à une technique d'imagerie non invasive ou à une thérapeutique qui interagit sélectivement avec le reporter ou le gène d'intérêt. Le sujet peut, par exemple, souffrir d'un cancer, et la thérapeutique peut être un agent anticancéreux. Le promoteur hTERT ou le promoteur hTERT amplifié peut entraîner l'expression d'un reporter, tel que hSSTR2 et/ou un dérivé de hSSTR2, ceux-ci pouvant être liés à un gène d'intérêt. L'imagerie peut être effectuée au moyen d'une variété de méthodes, notamment, la médecine nucléaire.
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WO2006099019A8 (fr) 2008-01-24
AU2006223435A1 (en) 2006-09-21
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RU2007137263A (ru) 2009-04-20

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