WO2008085564A2 - Composés et procédés impliquant des récepteurs de recombinaison tronqués couplés à la protéine g(7) - Google Patents

Composés et procédés impliquant des récepteurs de recombinaison tronqués couplés à la protéine g(7) Download PDF

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WO2008085564A2
WO2008085564A2 PCT/US2007/079092 US2007079092W WO2008085564A2 WO 2008085564 A2 WO2008085564 A2 WO 2008085564A2 US 2007079092 W US2007079092 W US 2007079092W WO 2008085564 A2 WO2008085564 A2 WO 2008085564A2
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promoter sequence
cell
disease
promoter
human
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WO2008085564A3 (fr
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Vikas Kundra
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University of Texas System
University of Texas at Austin
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    • 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
    • C07K14/72—Receptors; Cell surface antigens; Cell surface determinants for hormones
    • C07K14/723—G protein coupled receptor, e.g. TSHR-thyrotropin-receptor, LH/hCG receptor, FSH receptor
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    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6881—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for tissue or cell typing, e.g. human leukocyte antigen [HLA] probes
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    • C07K2319/00—Fusion polypeptide
    • C07K2319/40—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation
    • C07K2319/42—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation containing a HA(hemagglutinin)-tag
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    • C12N2799/00—Uses of viruses
    • C12N2799/02—Uses of viruses as vector
    • C12N2799/021—Uses of viruses as vector for the expression of a heterologous nucleic acid
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    • C12N2840/20—Vectors comprising a special translation-regulating system translation of more than one cistron
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Definitions

  • the present invention relates generally to the fields of cell biology, imaging, and molecular biology. More particularly, one aspect of the invention pertains to a method for tracking the location of a cell in a subject.
  • the cell is a stem cell or an immune cell.
  • the invention also generally pertains to methods for detecting the differentiation of a cell, such as a stem cell or an immune cell, in a subject.
  • Stem cells are cells that have the ability to continually reproduce themselves while maintaining the capacity to give rise to other more specialized types of cells. They may be obtained from natural sources, or generated through artificial means such as nuclear transfer, cytoplasmic transfer, cell fusion, parthenogenesis and reprogramming. Isolated stem cells can give rise to many types of differentiated cells. Two main groups of stem cells include adult stem cells and embryonic stem cells.
  • Adult stem cells are undifferentiated, but are capable of differentiation into the cell types from the tissue that the adult stem cell originated.
  • exemplary sources of adult stem cells include the nervous system (McKay, 1997; Shihabuddin et al, 1999), bone marrow (Pittenger et al, 1999; Pittenger and Marshak, 2001), adipose tissue (Gronthos et al, 2001), dermis (Toma et al, 2001), pancreas, liver (Deutsch et al, 2001), umbilical cord (Rogers et al, 2004; Wang et al, 2004; Surbek et al, 2002), and placenta (Yen et al, 2005).
  • stem cells of the adult type are also found in many other tissues, such as smooth muscle tissue, striated muscle tissue, cardiac muscle tissue, bone tissue, bone spongy tissue, cartilage tissue, pancreatic ductal tissue, spleen tissue, thymus tissue, tonsil tissue, Peyer's patch tissue, lymph nodes tissue, thyroid tissue, epidermis tissue, dermis tissue, subcutaneous tissue, heart tissue, lung tissue, vascular tissue, endothelial tissue, blood cells, bladder tissue, kidney tissue, digestive tract tissue, esophagus tissue, stomach tissue, small intestine tissue, large intestine tissue, adipose tissue, uterus tissue, eye tissue, lung tissue, testicular tissue, ovarian tissue, prostate tissue, connective tissue, endocrine tissue, and mesentery tissue.
  • tissues such as smooth muscle tissue, striated muscle tissue, cardiac muscle tissue, bone tissue, bone spongy tissue, cartilage tissue, pancreatic ductal tissue, spleen tissue, thymus tissue, tonsil
  • Stem cells can be applied in the treatment of many diseases.
  • stem cells derived from bone marrow cells are an established therapy in patients with hematological malignancies.
  • Stem cells have been utilized in the treatment of solid tumors.
  • Stem cells have also been used in "reprogramming" the immune system to induce an antigen-specific state of non-responsiveness called tolerance.
  • stem cells have been used to induce donor- specific tolerance during allogeneic or xenogeneic transplantation, and to induce tolerance in situations of autoimmunity.
  • stem cells One of the main therapeutic uses for stem cells is in the area of regenerative medicine.
  • the concept of regenerative medicine is to restore or enhance the ability of tissues to self- organize and heal themselves following endogenous or exogenous injury.
  • bone marrow-derived cells give rise to both hematopoietic cells and stroma cells.
  • Bone marrow cells have been shown to generate muscle fibers (Ferrari et ah, 1998), cardiomyocytes (Orlic et ah, 2001), microglia and astroglia (Eglitis and Mezey, 1997), and other tissues.
  • Stem cells have also been applied in the treatment of neurological deficiencies in a variety of situations. For example, administration of fetal stem cells into the striatal area of
  • Parkinson's disease patients results in the generation of dopaminergic neurons that can reinnervate the striatum, restore regulated dopamine release and movement-related frontal cortical activation, and result in observable clinical benefit (Lindvall et al, 2004).
  • immune cells include immune cells.
  • immune cells have been applied in the treatment of cancer.
  • An immune cell is defined herein to refer to a cell that recognizes and responds against microorganisms, viruses, and substances recognized as foreign and potentially harmful to the body. Examples of immune cells include T cells and B cells.
  • EBV Epstein-Barr virus
  • HSV-TK herpes simplex virus- 1 thymidine kinase
  • Dubey et al. (2003) demonstrated antigen specific localization of T cells expressing HSV-TK to tumors induced by murine sarcoma virus/Moloney murine leukemia virus (M-MS V/M-MuL V).
  • HSV-TK is not an optimal alternative for following stem cell or immune cell trafficking because it is known to induce an immune reaction (Berger et al, 2006; Perez-Cruet et al, 1994; Ramesh et al, 1996; Rainov et al, 2000).
  • the sodium/iodide symporter (NIS) has been used to follow transplantation of myoblasts (Vadysirisack et al, 2006).
  • the degree of NIS expression is not the sole reason for dictating the degree of radioiodide uptake, and the degree of sodium iodide uptake is saturable and therefore not linear beyond a certain degree of expression; further the maximal radioiodide uptake induced by NIS gene transfer differs among different cells (Vadysirisack et al, 2006).
  • GPCRs Seven G protein-coupled receptors
  • 7TM receptors seven transmembrane receptors
  • heptahelical receptors seven transmembrane receptors
  • GPCR family members are involved in all types of stimulus-response pathways. The diversity of functions is highlighted by the wide range of ligands recognized GPCRs, from photons (rhodopsin) to small molecules (histamine receptors) to proteins (chemokine receptors).
  • Somatostatin receptor belongs to the family of G protein-coupled receptors with seven transmembrane domains. SSTR2 can serve as a reporter of gene expression that can be quantified in vivo (Yang et al, 2005). Somatostatin receptors are over-expressed on a variety of tumors (John et al, 1996), and somatostatin receptor imaging can identify a variety of neuroendocrine malignancies, including carcinoid, islet cell tumor, pheochromocytoma, paraganglioma, small-cell lung cancer, and medullary thyroid cancer (Termanini et al, 1997; Lamberts et al., 2001; Kwekkeboom et al, 2000).
  • somatostatin For imaging, radiopharmaceutical analogs of the naturally occurring ligand, somatostatin, are used. Upon activation, somatostatin initiates a variety of signaling events that affect cellular functions such as secretion, chemotaxis, and growth suppression. These affects have been exploited using therapeutic analogs of somatostatin, for example, to ameliorate or prevent carcinoid syndrome (Nikou et al., 2005; Ducreux et al., 2000; Wymenga et al., 1999).
  • Types 2A and 2B are alternate splice variants that are identical, except that type 2A has a longer intracytoplasmic carboxy-terminus (Petersenn et al., 1999).
  • SSTR2 regulates signaling such as cAMP (Schwartkop et al., 1999) and cGMP production. The latter appears to regulate cell proliferation (Lopez et al., 2001).
  • SSTR2 can be localized and quantified in vivo using 111-Indium- labeled octreotide (Yang et al., 2005).
  • the receptor itself may be assessed. Signaling by the somatostatin receptors may be mediated through the C-terminus of the receptor (Schwartkop et al., 1999; Hukovic et al., 1998).
  • GPCRs such as SSTR2
  • SSTR2 for following the trafficking, differentiation, or localization of stem cells or immune cells has not been previously described.
  • GPCR's such as SSTR2 that are limited in signaling and in affecting phenotypic change have not been described for following trafficking, viability, trans/differentiation or fusion, localization, or expression of linked gene products of/by cells and their progeny in vivo using non-invasive or invasive imaging.
  • stem cells may engraft transiently into a diseased organ, engraft and not differentiate, or engraft and differentiate.
  • stem cells may engraft transiently into a diseased organ, engraft and not differentiate, or engraft and differentiate.
  • the inventor has identified novel methods of following the trafficking and differentiation of cells, such as stem cells or immune cells, in a subject. These methods involve obtaining a cell and transferring into the cell a nucleic acid that encodes a full length or truncated recombinant GPCR, or a GPCR amino acid sequence. Further, the inventor has found that truncation of the recombinant GPCR results in a receptor that can bind a ligand, but which is signaling defective and/or has altered internalization. This feature allows for the imaging of cells and limits effects on cellular signaling, function, or differentiation.
  • the present invention generally pertains to a method for tracking the location of a cell in a subject, involving: (a) obtaining a cell; (b) transferring into the cell an expression construct that includes a first coding region encoding a first reporter that includes a truncated recombinant seven transmembrane G-protein associated receptor (GPCR) amino acid sequence operatively linked to a first promoter sequence; (c) contacting the cell with a detectable moiety that binds to the first reporter; (d) introducing the cell to the subject; and (e) imaging the detectable moiety using an imaging technique.
  • GPCR seven transmembrane G-protein associated receptor
  • the cells are contacted with a detectable moiety after the cells have been introduced into a subject.
  • the method for tracking the location of a cell in a subject can be further defined as a method for tracking the location of progeny of the cell in a subject. For example, following transfer into a stem cell of an expression construct that encodes a truncated recombinant GPCR amino acid sequence, the expression construct becomes incorporated into the genome of the stem cell, and therefore the progeny of the stem cell would include a nucleic acid sequence that encodes the same truncated recombinant GPCR amino acid sequence.
  • progeny of the stem cell can be detected and imaged using any of the methods set forth herein.
  • the method may further involve contacting progeny of the cell with a detectable moiety that binds to the first reporter that is encoded in the progeny.
  • the cell that is obtained can be any cell known to those of ordinary skill in the art, but in particular embodiments the cell is a stem cell or an immune cell.
  • a “stem cell” generally refers to any cell that has the ability to divide for indefinite periods of time and to give rise to specialized cells.
  • the stem cell can be an embryonic stem cell, a somatic stem cell, a germ stem cell, an epidermal stem cell, or a tissue-specific stem cell.
  • tissue-specific stem cells include a cancer stem cell, an adult neural stem cell, a human neuron, a human oligodendrocyte, a human astrocyte, a human keratinocyte stem cell, a human keratinocyte transient amplifying cell, a human melanocyte stem cell, a human melanocyte, a human foreskin fibroblast, a human duct cell, a human pancreatic islet, a human pancreatic ⁇ -cell, a human adult renal stem cell, a human embryonic renal epithelial stem cell, a human kidney epithelial cell, a human hepatic oval cell, a human hepatocytes, a human bile duct epithelial cell, a human embryonic endodermal stem cell, a human adult hepatocyte stem cell (controvers
  • an "immune cell” is any cell associated with generation of an immune response, such as a monocyte, a granulocyte, or a lymphocyte.
  • the granulocyte may be a neutrophil, a basophil, or an eosinophil.
  • the lymphocyte may be a T cell, a B cell, or a NK cell.
  • the immune cell may be a stem cell whose progeny includes any of the aforementioned cells associated with generation of an immune response.
  • the cell can be obtained from any source, both natural and artificial.
  • the cell is an autologous cell.
  • the cell may be a stem cell obtained from a subject, wherein the cell is reintroduced into the subject following the transfer into the cell of the expression construct that comprises a coding region encoding a truncated recombinant GPCR.
  • the cell is an allogeneic cell, or a cell obtained from a subject that is distinct from the subject to whom the cell is introduced, but from the same species.
  • the cell is a xenogeneic stem cell, or a cell from a different species than the recipient subject.
  • the first reporter includes a truncated recombinant GPCR amino acid sequence.
  • the GPCR can be truncated at either the N- terminus or the C-terminus. In some embodiments, there is a truncation at both the N- terminus and the C-terminus.
  • the recombinant GPCR has a C- terminal deletion.
  • the truncation of the GPCR results in a GPCR that has altered signaling including is signaling defective, has altered internalization, or a combination thereof. Altered signaling includes an increase in signaling, a decrease in signaling, or an inciting of signaling pathways different from those incited by the wild type receptor.
  • the GPCR can be any GPCR known to those of ordinary skill in the art.
  • 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, CX
  • the GPCR is a somatostatin receptor.
  • the somatostatin receptor may be a somatostatin receptor type 1, 2A and 2B, 3, 4, or 5.
  • the somatostatin receptor is a somatostatin receptor type 2A (SSTR2A).
  • SSTR2A may have altered signaling including signaling defective, have altered internalization, or a combination thereof.
  • Information regarding somatostatin fusion proteins can be found in U.S. Patent App. Pub. No. 20020173626, herein specifically incorporated by reference.
  • the first promoter sequence can be any promoter sequence known to those of ordinary skill in the art. Promoter sequences are discussed in detail elsewhere in this specification.
  • the promoter sequence may be a function-specific promoter sequence, a constitutive promoter sequence, or a tissue-selective promoter sequence.
  • the first promoter sequence is a function-specific promoter sequence.
  • a "function specific promoter sequence" is a control sequence that is a region of a nucleic acid sequence at which initiation and rate of transcription are controlled, wherein the sequence is active in cells and whose products perform a particular function of interest.
  • tissue selective promoter sequences include an insulin promoter sequence, T cell receptor promoter sequence, immunoglobulin promoter sequence, hormone or paracrine promoters such as vascular endothelial growth factor promoter sequences, structural protein promoters such as a dystrophin promoter sequence, intracellular component such as fat or melanin promoter sequences, or extracellular component such as cartilage promoter sequences.
  • Other examples include a pBROAD promoter sequence, a c-fos promoter sequence, a c-HA-ras promoter sequence, an intercellular adhesion molecule 2 promoter sequence, and a platelet-derived growth factor (PDGF) promoter sequence.
  • PDGF platelet-derived growth factor
  • the first promoter sequence may also be a constitutive promoter sequence.
  • a "constitutive promoter sequence” is defined herein to refer to a control sequence that is a region of a nucleic acid sequence at which initiation and rate of transcription are controlled, wherein the sequence is active in cells of most any lineage.
  • the constitutive promoter sequence may be a beta-actin promoter sequence, an elastase I promoter sequence, a metallothionein (MTII) promoter sequence, a 5 S ribosomal promoter sequence, an Elastase promoter sequence, an Elastase I promoter sequence, a polyoma promoter sequence, a Cytomegalovirus promoter sequence, a retrovirus promoter sequence, a papilloma virus promoter sequence, a fibronectin promoter sequence, a ubiquitin promoter, an actin promoter, an elongation factor 1 alpha, an early growth factor response 1 , an eukaryotic initiation factor 4Al, a ferritin heavy chain, a ferritin light chain, a glyceraldehyde 3 -phosphate dehydrogenase, a glucose-regulated protein 78, a glucose-regulated protein 94, a heat shock protein 70, a heat shock protein 90, a beta beta
  • the constitutive promoter is a minimal viral promoter sequence.
  • the minimal viral promoter sequence may be a RNA virus promoter, DNA virus promoter, adenoviral promoter sequence, a baculoviral promoter sequence, a CMV promoter sequence, a parvovirus promoter sequence, a herpesvirus promoter sequence, a poxvirus promoter sequence, an adeno-associated virus promoter sequence, a semiliki forest virus promoter sequence, an SV40 promoter sequence, a vaccinia virus promoter sequence, a lentivirus promoter, or a retrovirus promoter sequence.
  • the first promoter sequence is a tissue selective promoter sequence.
  • tissue selective promoter sequence is defined herein to refer to a control sequence that is a region of a nucleic acid sequence at which initiation and rate of transcription are controlled, wherein the sequence is active in cells of a particular lineage or tissue type.
  • the tissue-selective promoter sequence may be a promoter sequence that is active in normal and/or diseased heart, lung, esophagus, muscle, intestine, breast, prostate, stomach, bladder, liver, spleen, pancreas, kidney, neurons, myocytes, leukocytes, immortalized cells, neoplastic cells, tumor cells, cancer cells, duodenum, jejunum, ileum, cecum, colon, rectum, salivary glands, gall bladder, urinary bladder, trachea, larynx, pharynx, aorta, arteries, capillaries, veins, thymus, lymph nodes, , bone marrow, pituitary gland, thyroid gland, parathyroid glands, adrenal glands, brain, cerebrum, cerebellum, medulla, pons, spinal cord, nerves, skeletal muscle, smooth muscle, bone, testes, epidiymides, prostate, seminal vesicles, pen
  • the tissue-selective promoter sequence is an hTR promoter sequence, a hTERT promoter sequence, a CEA promoter sequence, a PSA promoter sequence promoter sequence, a probasin promoter sequence, a ARR2PB promoter sequence, an AFP promoter sequence, a MUC-I promoter sequence, a MUC-4 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, a survivin promoter sequence, a Tcfl -alpha promoter sequence, a Ras promoter sequence, a Raf promoter sequence, a cyclin E promoter sequence, a Cdc25A promoter sequence, a HK II promoter sequence, a
  • tissue-selective promoter sequences include an immunoglobulin heavy chain promoter sequence, an immunoglobulin light chain promoter sequence, a T-cell receptor promoter sequence, an HLA DQ a promoter sequence, an HLA DQ beta promoter sequence, a beta-interferon promoter sequence, an interleukin-2 promoter sequence, an interleukin-2 receptor promoter sequence, an MHC Class II 5 promoter sequence, an MHC Class II HLA-Dra promoter sequence, , a muscle creatine kinase (MCK) promoter sequence, a prealbumin (transthyretin) promoter sequence, an albumin promoter sequence, an alpha- fetoprotein promoter sequence, a gamma-globin promoter sequence, a beta-globin promoter sequence, a, an insulin promoter sequence, a neural cell adhesion molecule (NCAM) promoter sequence, an alpha- 1 -antitrypsin promoter sequence, a growth hormone promoter sequence, a human
  • H2B histone promoter sequence
  • a type I collagen promoter sequence a GRP94 promoter sequence, a GRP78 promoter sequence, an other glucose-regulated protein promoter sequence, a Human Immunodeficiency Virus promoter sequence, a human LIMK2 gene promoter sequence, a murine epididymal retinoic acid-binding gene promoter sequence, a mouse alpha2 (XI) collagen promoter sequence, a DlA dopamine receptor promoter sequence, an insulin-like growth factor II promoter sequence, a human platelet endothelial cell adhesion molecule- 1 promoter sequence, a 7SL promoter sequence, a human MRP-7-2 promoter sequence, a leukosialin promoter sequence, a Sialophorin promoter sequence, a Macrosialin or human analogue of macrosialin promoter sequence, and an Endoglin promoter sequence.
  • the nucleic acid includes more than one coding region.
  • the nucleic acid may include a second coding region.
  • a second promoter sequence is operatively linked to the second coding region.
  • the first coding region and the second coding region are linked by an IRES or a bidirectional promoter sequence.
  • a "bidirectional promoter sequence" refers to control sequence that is a region of a nucleic acid sequence at which initiation and rate of transcription of both the first and the second coding region are controlled.
  • One of ordinary skill in the art would be familiar with bidirectional promoter sequences, such as those set forth in Trinklein et ah,
  • the first promoter sequence and the second promoter sequence may be promoter sequences that are individually selected from the group consisting of a constitutive promoter sequence, a tissue-specific promoter sequence, a lineage-specific promoter, and a function- specific promoter sequence.
  • the first promoter sequence and the second promoter sequence may be of the same type ⁇ e.g., both constitutive promoter sequences) or may differ in type ⁇ e.g., first promoter sequence is a constitutive promoter sequence, and second promoter sequence is a tissue-specific promoter sequence).
  • the second coding region may encode, for example, a reporter sequence, a therapeutic gene, or signaling sequence, or a trafficking sequence.
  • reporter for example, a reporter sequence, a therapeutic gene, or signaling sequence, or a trafficking sequence.
  • 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.
  • a "therapeutic gene” as used herein refers to any genetic sequence or encoding polynucleotide sequence that is known or suspected to be of benefit in the treatment or prevention of disease in a subject.
  • a “signaling sequence” is defined herein to refer to any genetic sequence or encoded polynucleotide sequence that is involved in signal transduction or cell differentiation.
  • a "trafficking sequence” as used herein refers to any genetic sequence or encoded polypeptide sequence that is involved in the transit of cells from one site in a subject to a different site in the subject.
  • the nucleic acid further includes a third coding region.
  • the third coding region is operatively linked to a third promoter sequence.
  • the first coding region, the second coding region, and the third coding region may either be independent or operably linked by one or more IRES or bidirectional promoter sequences.
  • the first promoter sequence, the second promoter sequence, and the third promoter sequence may be individually selected from the group consisting of a constitutive promoter sequence, a tissue-specific promoter sequence, a lineage-specific promoter, and a function-specific promoter sequence.
  • the promoter sequences may be of the same type or be of distinct types.
  • the first coding region, the second coding region, and the third coding region are individually selected from the group consisting of a reporter sequence, a therapeutic gene, or signaling sequence, or a trafficking sequence.
  • the first promoter sequence is a constitutive promoter sequence
  • the second promoter sequence is a tissue-specific promoter sequence
  • the third promoter sequence is a lineage specific promoter sequence or a function-specific promoter sequence.
  • the method further comprises sorting of the cell from other cells following the transfer of the expression construct.
  • Sorting refers to separation of a cell containing the expression construct from other cells that do not contain the expression construct. Sorting can be performed by any method known to those of ordinary skill in the art, and may rely on the presence of the encoded truncated recombinant GPCR. For example, sorting may be performed by fluorescence activated cell sorting (FACS), column chromatography, and/or magnetic resonance beads.
  • FACS fluorescence activated cell sorting
  • introducing the cell to the subject can be by any method known to those of ordinary skill in the art.
  • introducing the cell to the subject may involve intravenous administration, intracardiac administration, intradermal administration, intralesional administration, intrathecal administration, intracranial administration, intrapericardial administration, intraumbilical administration, intraocular administration, intraarterial administration, intraperitoneal administration, intraosseous administration, intrahemmorhage administration, intratrauma administration, intratumor administration, subcutaneous administration, intramuscular administration, intravitreous administration, direct injection into a normal organ, direct injection into a diseased organ, topical administration, or any other method of local or systemic administration known to those of ordinary skill in the art.
  • the method for tracking the location of a cell in a subject further comprises detecting expression of the first reporter by assaying for an association between the reporter expressed by the cell and a detectable moiety.
  • the association between the cell and the detectable moiety comprises binding of the detectable moiety by the cell, binding of a ligand operably coupled to the detectable moiety by the cell, cellular uptake of the detectable moiety, or cellular uptake of a ligand operably coupled to the detectable moiety.
  • a “detectable moiety” is defined herein to refer to any molecule or agent that can emit a signal that is detectable by imaging.
  • the detectable moiety may be a protein, a radioisotope, a fluorophore, a visible light emitting fluorophore, a near infrared light emitting fluorophore, infrared light emitting fluorophore, a metal, a ferromagnetic substance, a paramagnetic substance, a superparamagnetic 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 a radioisotope.
  • the detectable moiety is 111- In octreotide.
  • the detectable moiety is operably coupled to a ligand that specifically binds the reporter.
  • a "ligand” is defined herein to refer to an ion, a peptide, a oligonucleotide, a molecule, or a molecular group that binds to another chemical entity or polypeptide to form a larger complex.
  • 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 detectable moiety is a small molecule.
  • the detectable moiety is a near infrared light-emitting fluorophore.
  • the nucleic acid further encodes a protein tag fused to the N- terminal end or C-terminal end of the truncated GPCR amino acid sequence.
  • the protein tag may or may not have enzymatic activity.
  • the protein tag may be, for example, hemagglutinin A, beta-galactosidase, thymidine kinase, transferrin, myc-tag, VP 16, (His) 6 -tag, FLAG, or chloramphenicol acetyl transferase.
  • the expression construct that is transferred into the cell may or may not be 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, lipofectamine, a plasmid, a viral vector, a phage, a polyamino acid such as polylysine, a prokaryotic cell, or a eukaryotic cell.
  • the expression construct is comprised in a delivery vehicle, and transferring the expression construct into the cell comprises contacting the cell with the delivery vehicle.
  • the delivery vehicle is a viral vector.
  • the viral vector can be any viral vector known to those of ordinary skill in the art.
  • the viral vector may be a lentiviral vector, a baculovirus vector, a parvovirus vector, a semiliki forest virus vector, a Sindbis virus vector, a lentivirus vector, a retroviral vector, a vaccinia viral vector, an adeno-associated viral vector, a picornavirus vecctor, an alphavirus vector, or a poxviral vector.
  • the viral vector is a lentiviral vector.
  • Transferring the expression construct into the cell can be by any method known to those of ordinary skill in the art.
  • transferring the expression construct may involve performing electroporation or nucleofection of the cell in the presence of the expression construct.
  • the cells are contacting with a detectable moiety that binds to the first reporter prior to introducing the cells into the subject, and in other embodiments, the cells are contacted with the detectable moiety that binds to the first reporter after the cells are introduced into the subject.
  • the imaging technique is an invasive imaging technique.
  • An "invasive imaging technique” is defined herein to refer to any imaging technique that involves removal of tissue from a subject or insertion of a medical device into a subject. Invasive imaging techniques may involve, for example, performance of a biopsy of tissue in conjunction with an imaging technique such as fluorescence microscopy, or insertion of a catheter or endoscope into a subject for purposes of imaging.
  • the imaging technique is a non-invasive imaging technique.
  • a "non-invasive imaging technique” is defined herein as an imaging technique that does not involve removal of tissue from a subject or insertion of a medical device into a subject.
  • One of ordinary skill in the art would be familiar with non-invasive imaging techniques. Examples 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, and imaging using infrared light.
  • the method for tracking the location of a cell in a subject may be further defined as a method for treating a subject with disease.
  • the cell to be tracked is a stem cell, and the stem cell is introduced into the subject for the purpose of treating a disease.
  • the disease can be any disease known to those of ordinary skill in the art.
  • the disease may be a hyperproliferative disease, an infectious disease, an inflammatory disease, a degenerative disease, a congenital disease, a genetic disease, an immunological disease, trauma, poisoning, or a disease associated with toxicity.
  • the disease is a hyperproliferative disease.
  • the hyperproliferative disease may be benign or malignant.
  • the hyperproliferative disease is cancer.
  • the cancer may be any type of cancer.
  • the cancer may be breast cancer, lung cancer, prostate cancer, ovarian cancer, brain cancer, liver cancer, cervical cancer, colon cancer, renal cancer, skin cancer, head and neck cancer, bone cancer, esophageal cancer, bladder cancer, uterine cancer, lymphatic cancer, stomach cancer, pancreatic cancer, testicular cancer, lymphoma, or leukemia.
  • the disease is type I diabetes or type II diabetes.
  • the disease is cardiovascular disease.
  • the cardiovascular disease may be cardiomyopathy, ischemic cardiac disease, congestive heart failure, congenital cardiac disease, traumatic cardiac disease, toxic cardiac disease, pericarditis, or genetic cardiac disease.
  • the disease may be a neurological disease, such as Parkinson's disease, Alzeimer disease, amyotrophic lateral sclerosis, or multiple sclerosis.
  • the neurological disease may be a neurodegenerative disease, spinal cord disease, traumatic neurological disease, infectious disease, or inflammatory disease.
  • the disease may also be an immunological disease, such as transplant rejection, autoimmune disease, immune complex disease, vasculitis, or HIV infection.
  • the method for tracking the location of a cell and/or its progeny in a subject is further defined as a method of assessing the viability of a cell and/or its progeny in a subject.
  • the expression construct may include a first coding region encoding a reporter comprising a truncated recombinant GPCR amino acid sequence operatively linked to a constitutive promoter sequence. Imaging a detectable moiety that binds to the truncated recombinant GPCR sequence would provide an indicator not only on location, but of cell viability.
  • the method for tracking the location of a cell in a subject may further be defined as a method for assessing the trans/differentiation or fusion of a stem cell and/or its progeny in a subject.
  • the expression construct may include a first coding region encoding a reporter comprising a truncated recombinant GPCR amino acid sequence operatively linked to a constitutive promoter sequence. Imaging a detectable moiety that binds to the truncated recombinant GPCR sequence would provide an indicator not only on location, but of cell viability.
  • Imaging of a second coding region encoding a reporter operatively coupled to a second promoter sequence would provide evidence of differentiation of a stem cell and/ or its progeny in the subject, as the second coding region would only be encoded under conditions associated with some extent of cell differentiation.
  • a second promoter sequence such as a tissue-selective promoter sequence, a lineage-specific promoter sequence, or a function-specific promoter sequence
  • the method for tracking the location of a cell in a subject is further defined as a method for tracking the location of a tissue that is transplanted into a subject.
  • the tissue can be any tissue suitable for transplantation.
  • the tissue may be heart tissue, islet cell tissue, or tissue from any organ that expresses a recombinant truncated GPCR.
  • introducing the cell into the subject is further defined as transplanting the tissue into the subject.
  • the transplanted tissue may comprise one or more cells that include a reporter encoding a truncated recombinant GPCR amino acid sequence.
  • the tissue may be made to differentiation into tissues of organs (e.g., embryonic stem cells into hear tissue) in vitro or in vivo, and then be transplanted into another animal.
  • the truncated GPCR reporter may be used, for example, to track viability, incorporation, and trafficking of cells that may migrate from the graft. This is important for tissue engineering, such as generation of a new external ear or liver.
  • One embodiment of the present invention is a method for tracking the location of a stem cell and/or its progeny in a human subject, which involves (a) obtaining a stem cell; (b) transfecting the stem cell with an expression construct comprising a first coding region encoding a first reporter comprising a recombinant somatostatin receptor truncated carboxy- terminal to amino acid 314 and operatively linked to a first promoter sequence; (c) introducing the stem cell to the subject; and (d) detecting the location of the stem cell and/or its progeny in the subject using an imaging technique to detect a detectable moiety that is bound to the truncated recombinant somatostatin receptor.
  • a further embodiment of the present invention is a method for detecting the differentiation of a stem cell and/or its progeny in a human subject, which involves: (a) obtaining a stem cell; (b) transfecting the stem cell with an expression construct comprising a first coding region encoding a first reporter comprising a somatostatin receptor truncated carboxy-terminal to amino acid 314 and operatively linked to a first promoter sequence; (c) introducing the stem cell to the subject; and (d) detecting the differentiation of the stem cell in the subject using an imaging technique to detect a detectable moiety that is bound to the truncated recombinant somatostatin receptor.
  • the stem cell can be any of those stem cells discussed above and elsewhere in this specification.
  • the stem cell is an immune progenitor cell.
  • An immune progenitor cell is any cell whose progeny is an immune cell, such as any of the immune cells discussed above.
  • a further embodiment pertains to a method of tracking the location, lineage differentiation and function of a stem cell.
  • the stem cells can be (a) tracked to the pancreas using a constitutive promoter, (b) evaluated for trans/differention into islet cell lineage using an islet cell promoter such as Nkx2.2, and (c) assessed for function using a functional promoter such as the insulin promoter.
  • one or multiple types of immune cells are used, such as T- lymphocytes that bind a particular antigen or peripheral blood white blood cells, respectively.
  • the present invention also generally pertains to non-human transgenic animals whose genomes comprise a nucleic acid encoding a truncated recombinant GPCR amino acid sequence.
  • Transgenic animals are non-human animals, preferably mammals, in which one or more of the cells include a transgene. Exemplary transgenic animals include primates, sheep, dogs, cats, rabbits, cows, goats, birds such as chickens, reptiles, amphibians, rodents such as rats and mice, etc. In certain embodiments, the transgenic animal is a mouse.
  • the present invention also generally pertains to non-human transgenic animals whose genome comprises a nucleic acid encoding a recombinant GPCR receptor under the control of a heterologous promoter.
  • a “heterologous promoter” is a promoter that is out of its naturally occurring context.
  • the GPCR can be any GPCR known to those of ordinary skill in the art, including those set forth above.
  • the GPCR is a somatostatin receptor, such as SSTR2.
  • the heterologous promoter may be a constitutive promoter, a tissue-selective promoter, a lineage-specific promoter sequence, or a functional promoter.
  • the transgenic animal is a primate such as a monkey.
  • a "transgene” is exogenous DNA that is integrated into the genome of a cell from which a transgenic animal develops, and that remains in the genome of the mature animal.
  • the nucleic acid encoding the truncated recombinant GPCR preferably is operably linked to a promoter.
  • the promoter may be any promoter sequence, including any of those promoter sequences discussed above and elsewhere in this specification.
  • a cell of the transgenic animal expresses a truncated recombinant seven transmembrane G- protein associated receptor (GPCR) amino acid sequence.
  • the truncated recombinant GPCR is a truncated somatostatin receptor.
  • the somatostatin receptor is a somatostatin receptor type 2A (SSTR2A) or a somatostatin receptor that is truncated carboxy terminal to amino acid 314.
  • the somatostatin receptor is a SSTR2delta314, which is a SSTR2A truncated carboxy terminal to amino acid 314.
  • the transgenic animal may be of any species, such as primates, sheep, dogs, cats, cows, goats, birds such as chickens, reptiles, amphibians, rodents such as rats and mice, etc.
  • the transgenic animal is a mouse.
  • the transgenic animal is a primate such as a monkey.
  • Techniques pertaining to transgenic animals are well-known to those of ordinary skill in the art, and are reviewed in the specification below. Aspects of the present invention also generally pertain to a method of producing a cell that expresses a truncated recombinant GPCR amino acid sequence, that involves obtaining any of the transgenic animals set forth above and isolating one or more cells from the transgenic animal.
  • the cell is a stem cell, an immune cell, or a cancer cell.
  • the stem cell can be any stem cell, including any of the stem cells discussed above.
  • the stem cell is an embryonic stem cell or a somatic stem cell.
  • the truncated recombinant GPCR is a truncated somatostatin receptor.
  • the somatostatin receptor is truncated carboxy terminal to amino acid 314.
  • the recombinant somatostatin receptor is a somatostatin receptor type 2A (SSTR2A).
  • the present invention also pertains to methods of producing a cell that expresses a recombinant somatostatin receptor amino acid sequence, comprising (a) obtaining a non- human transgenic animal whose genome includes a nucleic acid encoding a first reporter that includes a somatostatin receptor amino acid sequence and (b) isolating one or more cells from said transgenic animal.
  • the somatostatin receptor amino acid sequence is a somatostatin receptor type 2A (SSTR2A) amino acid sequence.
  • inventions of the present invention pertain to methods for tracking the location of a cell in a subject, that involve (a) contacting a cell produced by any of the methods set forth herein with a detectable moiety that binds to the first reporter; (b) introducing the cell to the subject; and (c) imaging the detectable moiety using an imaging technique.
  • the nucleic acid encoding the truncated recombinant GPCR amino acid sequence or SSTR amino acid sequence preferably is operably linked to a promoter.
  • the promoter may be any promoter sequence, including any of those promoter sequences discussed above and elsewhere in this specification.
  • the promoter may be a constitutive promoter, a tissue-selective promoter, a lineage-specific promoter sequence, or a functional promoter sequence.
  • the nucleic acid encoding the truncated recombinant GPCR amino acid sequence may or may not further comprise a nucleic acid sequence encoding a protein tag.
  • the protein tag may be any protein tag known to those of ordinary skill in the art. Examples are discussed elsewhere in this specification.
  • isolating the one or more cells from the transgenic animal involves sorting the cells. Sorting is performed to separate a cell expressing the encoded truncated recombinant GPCR from a cell that does not express the truncated recombinant GPCR. Sorting can be performed by any method known to those of ordinary skill in the art, including any of those methods discussed above and elsewhere in this specification. For example, sorting may involve FACS, separation using magnetic resonance beads, and/or column chromatography.
  • tissues such as islets and organs that express the truncated GPCR reporter that are obtained from a transgenic animal may be transplanted into a subject, and the reporter may be used, for example, to track the viability, incorporation, and trafficking of cells that may migrate from the graft.
  • stem cells expressing the reporter may be made to differentiate into tissues or organs (e.g., embryonic stem cells into heart tissue) in vitro or in vivo, and then be transplanted into another animal.
  • the reporter may be used, for example, to track viability, incorporation, and trafficking of cells that may migrate from the graft. This is important for tissue engineering, such as generation of a new external ear or liver.
  • the reporter may be a GPCR such as SSTR, including SSTR2, or a GPCR with altered signaling and/or internalization such as a truncated SSTR reporter, including a truncated SSTR2 reporter.
  • the method is further defined as a method of treating a subject.
  • the subject may have any disease, such as any of the diseases discussed above.
  • composition of the invention may be used in any method of the invention, and any method of the invention may be used to produce or to utilize any composition of the invention.
  • the words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), "including” (and any form of including, such as “includes” and “include”) or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.
  • FIGS. IA-I Cell lines transfected with HA-wt SSTR2 or HA-SSTR2 ⁇ 314 express appropriate transcripts and similar levels of fusion protein.
  • FIG. IA Untransfected HT 1080 or HEK 293 cells do not express somatostatin receptors. No bands are seen in lanes SSTRl -5 of HT 1080 or HEK293 representing RT-PCR of cellular RNA. The quality of the same RNA for RT-PCR is confirmed by bands in the ⁇ -actin lanes (blank, no RNA). Lanes SSTRcDNA
  • FIGS. IB-C Transcripts of the appropriate size are detected by reverse-transcriptase polymerase chain reaction in HT1080 cells, as in FIG.
  • FIGS. ID-G Immunofluorescence targeting the HA domain demonstrate cell membrane localization of HA-wt or HA-SSTR2 ⁇ 314 fusion proteins; HT1080 clone expressing HA-wt, as shown in FIG. ID, or HA-SSTR2 ⁇ 314, as shown in FIG. IE, HEK293 clone expressing HA-wt, as shown in FIG. IF, or HA-SSTR2 ⁇ 314, as shown in FIG.
  • FIGS. IH-I Equal protein expression is detected by ELISA targeting the HA domain in HT 1080, as shown in FIG. IH, or HEK 293, as shown in FIG. II, cell line pairs transfected with HA-wt or HA-SSTR2 ⁇ 314; whereas, no expression is seen in cells transfected with vector. Error bars represent SD of triplicate samples (*P ⁇ 0.05, Vector vs HA-wt SSTR2 or HA-SSTR2 ⁇ 314).
  • FIGS. 2A-E HA-wild-type SSTR2 and HA-SSTR2 ⁇ 314 demonstrated similar binding to " ⁇ -octreotide.
  • FIG. 2A, FIG. 2B, FIG. 2C, FIG. 2D Representative Scatchard (left) and saturation (right) plots of 111 In-octreotide binding to membranes from HT 1080 cells transfected with HA-wt SSTR2, as in FIG. 2A, or HA-SSTR2 ⁇ 314, as in FIG. 2B, and of HEK293 cells transfected with HA-wt SSTR2, as in FIG. 2C, or HA-SSTR2 ⁇ 314, as in FIG. 2D.
  • FIG. 2E The left graph in FIG. 2E shows the uptake of ul In-octreotide by cells in vitro in the presence or absence of unlabeled somatostatin by HT1080 clones.
  • the right graph in FIG. 2E shows the uptake of l u In-octreotide by cells in vitro in the presence or absence of unlabeled somatostatin by HEK 293 clones.
  • the abbreviation "ss” refers to somatostatin; "V” refers to vector.
  • FIGS. 3A-D HA-SSTR2 ⁇ 314 is signaling deficient for the cAMP pathway.
  • FIG. 3A and FIG. 3C Upon ligand binding, HA-wt SSTR2 decreases forskolin induced cAMP production in HT 1080 cells, as shown in FIG. 3 A, or in HEK 293 cells, as shown in FIG. 3C. *P ⁇ 0.05.
  • FIG. 3B and FIG. 3D Upon ligand binding, HA-SSTR2 ⁇ 314 does not decreases forskolin induced cAMP production when expressed in HT 1080 cells, as shown in FIG. 3B, or in HEK 293 cells, as shown in FIG. 3D.
  • FIGS. 4A-D HA-SSTR2 ⁇ 314 is signaling deficient for the cGMP pathway.
  • FIG. 4A and FIG. 4C Upon ligand binding, HA-wt SSTR2 incites cGMP production when expressed in HT1080 cells, as shown in FIG. 4A, or in HEK 293 cells, as shown in FIG. 4C. (*P ⁇ 0.05, Phosphate-buffered saline (PBS) vs SS).
  • FIG. 4B and FIG. 4D Upon ligand binding, HA- SSTR2 ⁇ 314 does not incite cGMP production when expressed in HT1080 cells, as shown in FIG.
  • FIGS. 5A-D HA-SSTR2 ⁇ 314 is deficient in inhibiting cell growth. Values on the abscissa are expressed as percent of color product of cells treated with Sandostatin compared to the same cells not exposed to Sandostatin.
  • FIG. 5 A and FIG. 5C Upon ligand binding, HA- wt SSTR2 decreases proliferation induced by serum in HT1080 cells, as shown in FIG. 5A, or in HEK 293 cells, as shown in FIG. 5C. *P ⁇ 0.05.
  • FIG. 5B and FIG. 5D Upon ligand binding, HA-SSTR2 ⁇ 314 does not decrease proliferation induced by serum in HT1080 cells, as shown in FIG. 5B, or in HEK 293 cells, as shown in FIG. 5D.
  • FIGS. 6A-B HA-wt SSTR2 and HA-SSTR2 ⁇ 314 are competent for imaging in vivo.
  • FIG. 6A Representative planar ⁇ -camera image of a nude mouse demonstrates that tumors derived from HT1080 cells expressing HA-wt SSTR2 (left shoulder) or expressing HA-SSTR2 ⁇ 314 (right shoulder) are visible, but the tumor derived from HT 1080 cells transfected with vector is not visible (left thigh).
  • FIG. 6B Nude mice bearing subcutaneous tumors were injected intravenously with l u In-octreotide (13 MBq) and imaged 24 hours later.
  • FIG. 7 Stably transfected HS-5 cells express similar amounts of HA-SSTR2 ⁇ 314 (A314) or HA-wt SSTR2 (wt). Quantitative ELISA using an antibody to the HA-domain demonstrates equal binding. See legend to FIG. 1. Error bars represent SD of triplicate samples. "mU” refers to milliunits of horse radish peroxidase enzymatic activity.
  • FIG. 8 Stably transfected HS-5 cells expressing HA-SSTR2 ⁇ 314 (A314) or HA-wt SSTR2 (wt) bind similar amounts of 111 In octreotide. Binding was performed using 10 "7 M 111 In octreotide and competition was performed using 10 "6 M somatostatin. Together with the the previous ELISA of the HS5 cells, these results show that the cells have equal fusion protein expression (ELISA targeting the HA tag) and equal binding to H l-In octreotide implying that the receptor mutation does not inhibit the ability of the receptor to bind the imaging ligand 1 H-In octreotide. This conclusion is also reached by IBand of FIGS. 2 A-D.
  • FIG. 9 HA-SSTR2 ⁇ 314 is signaling deficient for the cAMP pathway in human bone marrow mesenchymal cells, HS-5. Upon ligand (100 nM somatostatin- 14) binding, HA-wt
  • SSTR2 decreases forskolin induced cAMP production (wt) whereas HA-SSTR2 ⁇ 314 ( ⁇ 314) does not when expressed in HS-5 cells (*P ⁇ 0.05).
  • HS-5 cells were stably transfected with HA-wt SSTR2 or HA-SSTR2 ⁇ 314.
  • FIG. 10 HA-SSTR2 ⁇ 314 is signaling deficient for the cGMP pathway in human bone marrow mesenchymal cells, HS-5. Upon ligand (100 nM somatostatin- 14) binding, HA-wt SSTR2 (wt) incites cGMP production, whereas HA-SSTR2 ⁇ 314 ( ⁇ 314) does not when expressed in HS-5 cells (*P ⁇ 0.05).
  • HA-SSTR2 ⁇ 314 is deficient in inhibiting cell growth in human bone marrow mesenchymal cells, HS-5.
  • HA-wt SSTR2 Upon ligand (100 nM Sandostatin) binding, HA-wt SSTR2 (wt) decreases proliferation induced by serum, whereas HA-SSTR2 ⁇ 314 ( ⁇ 314) does not when expressed in HS-5 cells (*P ⁇ 0.05).
  • Values on the abscissa are expressed as percent of color product of cells treated with Sandostatin compared to the same cells not exposed to Sandostatin using a MTT assay.
  • FIG. 12 Human bone marrow mesenchymal cells, HS-5, expressing HA-SSTR2 ⁇ 314 differentiate into osteoclast lineage cells that produce calcium phosphate.
  • osteoclast differentiation medium calcium phosphate production was equivalent in cells transfected with vector or HA- SSTR2 ⁇ 314, but decreased in cells transfected HA-wt SSTR2. (*P ⁇ 0.05).
  • FIGS. 13A-F Human bone marrow mesenchymal cells, HS-5, expressing HA-SSTR2 ⁇ 314 differentiate into adipocyte lineage.
  • HS- 5. expressing HA-SSTR2 ⁇ 314.
  • HS-5 cells stably transfected with (A) HA-SSTR2 ⁇ 314, (B) HA-wt SSTR2, or (C) vector were exposed to 10 "7 M 1 H-In octreotide for two hours, washed and then injected into C57/B16 mice via tail vein.
  • D No cell control incubated with 10 "7 M H l-In octreotide for two hours, washed and then injected into C57/B16 mice via tail vein.
  • SSTR2 ⁇ 314 is signaling deficient for the cAMP pathway in human bone marrow mesenchymal cells, H S -5.
  • ligand 100 nM somatostatin- 14 binding
  • wild type SSTR2 decreases forskolin-induced cAMP production (wt)
  • SSTR2 ⁇ 314 ⁇ 3114 does not when expressed in HS-5 cells (*P ⁇ 0.05, Forskolin vs. Fors+SS).
  • HS-5 cells were stably transfected with wt SSTR2 or SSTR2 ⁇ 314.
  • FIG. 16 SSTR2 ⁇ 314 is signaling deficient for the cGMP pathway in human bone marrow mesenchymal cells, HS-5. Upon ligand (100 nm somatostatin- 14) binding, wt SSTR2 (wt) incites cGMP production, whereas SSTR2 ⁇ 314 ( ⁇ 314) does not when expressed in HS-5 cells (*P ⁇ 0.05, PBS vs. SS 14).
  • FIG. 17 SSTR2 ⁇ 314 is deficient in inhibiting cell growth in human bone marrow mesenchymal cells, HS-5.
  • wt SSTR2 decreases proliferation induced by serum, whereas SSTR2 ⁇ 314 ( ⁇ 314) does not when expressed in HS-5 cells (*P ⁇ 0.05). Values on the abscissa are expressed as percent of color product of cells treated with Sandostatin compared to the same cells not exposed to Sandostatin using a MTT assay.
  • FIG. 18 Human bone marrow mesenchymal cells, HS-5, expressing SSTR2 ⁇ 314 differentiate into osteoclast lineage cells that produce calcium phosphate.
  • Cells transfected with vector, SSTR2 ⁇ 314 ( ⁇ 314), or wt SSTR2 (wt) produced calcium phosphate upon exposure to osteogenic induction medium.
  • osteoclast differentiation medium calcium phosphate production was equivalent in cells transfected with vector or SSTR2 ⁇ 314, but decreased in cells transfected with wt SSTR2. (*P ⁇ 0.05, vector vs. wt SSTR2).
  • FIG. 19 Human bone marrow mesenchymal cells, HS-5, expressing SSTR2 ⁇ 314 differentiate into adipocyte lineage. Cells transfected with vector (upper left, upper right), wt SSTR2 (middle left, middle right), or SSTR2 ⁇ 314 (lower left, lower right) demonstrate increased staining for fat using Oil Red O with exposure to adipogenic induction medium (upper right, middle right, lower right) compared to without exposure (upper left, middle left, lower left).
  • Oil Red O Oil Red O
  • adipogenic induction medium upper right, middle right, lower right
  • Magnification 400 X Magnification 400 X.
  • FIG. 20A-C In vivo imaging of mesenchymal stem cells, HS5 cells expressing HA-SSTR2 or HA-SSTR2 ⁇ 314 (FIG. 20A) or SSTR2 or SSTR2 ⁇ 314 (FIG. 20B), coiniected with ovarian cancer cells to differentiate and serve as stromal support for the ovarian tumor. Expression of either HA-SSTR2 (short arrow) or HA- SSTR2 ⁇ 314 (long arrow) is visualized in FIG. 2OA or SSTR2 (short arrow) or SSTR2 ⁇ 314 (long arrow) is visualized in FIG. 2OB. Negative controls, HS5 cells transfected with vector (arrowhead), are not seen in FIG. 2OA or B.
  • Stably transfected HS5 cells were coinjected with ovarian cancer cells (HeyA8) and after tumor formation, the mice were injected with 300 microCuries of H l-In octreotide via tail vein. Planar imaging using a gamma camera was performed the next day.
  • FIG. 20C Biodistribution. Increased uptake is seen in tumors incorporating HS5 cells expressing HA- SSTR2, HA- SSTR2 ⁇ 314, SSTR2, or SSTR2 ⁇ 314 compared to tumors incorporating HS5 cells expressing vector (P ⁇ 0.05). No statistically significant difference is seen among tumors incorporating HS5 cells expressing HA-SSTR2, HA- SSTR2 ⁇ 314, SSTR2 or SSTR2 ⁇ 314.
  • FIG. 21 Isolated human peripheral blood white blood cells. Human peripheral white blood cells isolated via a Ficoll gradient and stained with Hematoxylin demonstrating nuclei. Magnification 400X
  • FIG. 22 Isolated human peripheral blood white blood cells infected with adenovirus containing an insert for HA-SSTR2 ⁇ 314 ( ⁇ 314) or HA- wt SSTR2 (wf) express the fusion proteins. Quantitative ELISA using an antibody to the HA-domain. Expression of HA-wt SSTR2 (wt) was greater than that of HA-SSTR2 ⁇ 314 ( ⁇ 314) in these sets of cells used for the cell trafficking experiment. P ⁇ 0.05.
  • FIG. 23 In vivo imaging of cell trafficking of isolated human peripheral white blood cells expressing HA-SSTR2 ⁇ 314.
  • White blood cells infected with adenovirus containing an insert for (A) HA-wt SSTR2, (B) HA-SSTR2 ⁇ 314, or (C) control were exposed to 10 "7 M H l-In octreotide for two hours, washed and then injected into nude mice via tail vein.
  • D No cell control incubated with 10 "7 M 111-In octreotide for two hours, washed and then injected into nude mice via tail vein.
  • Expression of HA-wt SSTR2 was greater than that of HA-SSTR2 ⁇ 314 in these sets of cells used for the cell trafficking experiment (see FIG. 22) and this is reflected in the imaging.
  • the planar gamma camera image was collected from 10 to 60 minutes post injection.
  • FIGS. 24A-C Example constructs incorporating HA-SSTR2 ⁇ 314. (Introns need not be used, but can be helpful to increase expression in transgenic animals and their positions in the construct may be varied.)
  • FIG. 24A Examples of constructs using constitutive promoters such as CMV and human Ubiquitin.
  • FIG. 24B A functional and tissue selective promoter to assess for activation of the albumin promoter and as a marker for hepatocytes, since albumin expression is essentially restricted to hepatocytes.
  • FIG. 24C Example of an amplified functional and tissue selective promoter (miniCMV) for amplifying expression from the albumin promoter.
  • miniCMV amplified functional and tissue selective promoter
  • FIG. 25 Transgenic mice express HA-SSTR2 ⁇ 314 mRNA in multiple organs. Reverse transcriptase-polymerase chain reaction of RNA derived from transgenic mice (Ml and M2) or non-transgenic parental strain mouse (P). Primers for HA-SSTR2 ⁇ 314 demonstrate expression of HA-SSTR ⁇ 314 in only transgenic mice. Primers for the ubiquitously expressed ⁇ -actin demonstrate loading of RNA. "MW marker” signifies a molecular weight marker. "Blank” signifies that no RNA was added to the reaction. Transgenic mice were created using a construct with an ubiquitin promoter for driving expression of HA-
  • FIG. 26 Transgenic mice express SSTR2 ⁇ 314 protein in multiple organs.
  • Western blot of protein derived from transgenic mice (Ml and M2) or non-transgenic parental strain mouse (P) demonstrate expression of HA-SSTR ⁇ 314 in only transgenic mice.
  • Bone marrow was combined from two different transgenic mice (T) to obtain enough protein for the Western blot.
  • Transgenic mice were created using a construct with an ubiquitin promoter for driving expression of HA-SSTR2. The primary antibody targeted the HA domain.
  • FIG. 27 Transgenic mice express HA-SSTR2 ⁇ 314 protein in the liver.
  • Western blot of protein derived from transgenic mouse (ALB) or non-transgenic parental strain mouse (wt) demonstrate expression of HA-SSTR2 ⁇ 314 in the liver of the transgenic mice.
  • Transgenic mice were created using a construct with a miniCMV-albumin promoter for driving expression of HA-SSTR2 ⁇ 314. The primary antibody targeted the HA domain.
  • a new form of imaging or molecular imaging that has developed during the past decade involves the 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).
  • the present invention provides for methods of imaging a cell and/or its progeny that involve transferring into the cell an expression construct encoding a reporter that includes a truncated recombinant GPCR amino acid sequence.
  • the inventors have found that certain truncated recombinant somatostatin receptor amino acid sequences are signaling defective and/or have altered internalization.
  • truncated recombinant somatostatin receptor in a stem cell has no significant effect on differentiation of the stem cell or on inhibition of cAMP and activation of cGMP production of the stem cell.
  • the present invention allows for the imaging of cells that contain modified somatostatin receptors without causing cAMP and cGMP signaling, or otherwise affecting cellular functions such as growth suppression. Furthermore, the inventors have found that the truncation of the C-terminus of the somatostatin receptors serves to uncouple imaging from signal transduction.
  • SSTR2 can function as a reporter of gene transfer, and expression can be imaged in vivo. It has been found that by using ⁇ indium-labeled octreotide and a C-terminus truncated SSTR2, in vivo SSTR2A imaging can be uncoupled from signal transduction.
  • the cells that are employed in the methods of the present invention can be any type of cell, such as a eukaryotic cell or a prokaryotic cell.
  • the cells are stem cells or immune cells, such as immune progenitor cells.
  • stem cell generally refers to any cells that have the ability to divide for indefinite periods of time and to give rise to specialized cells.
  • the definition of “stem cell” includes, but is not limited to: a) totipotent cells such as an embryonic stem cell, an extraembryonic stem cell, a cloned stem cell, a parthenogenesis derived cell, a cell reprogrammed to possess totipotent properties, or a primordial germ cell; b) pluripotent cell such as a hematopoietic stem cell, an adipose derived stem cell, a mesenchymal stem cell, a cord blood stem cell, a placentally derived stem cell, an exfoliated tooth derived stem cells, a hair follicle stem cell or a neural stem cell; and c) a tissue specific progenitor cell such as a precursor cell for the neuronal, hepatic, nephrogenic, adipogenic, osteoblastic, osteoclastic, al
  • the cells that are employed in the methods of the present invention can be obtained from any source known to those of ordinary skill in the art.
  • the cells are stem cells obtained from a donor.
  • the cells are obtained from the subject who is to receive the cells as part of a therapeutic procedure.
  • the cells can be derived, for example, from tissues such as pancreatic tissue, liver tissue, smooth muscle tissue, striated muscle tissue, cardiac muscle tissue, bone tissue, bone marrow tissue, bone spongy tissue, cartilage tissue, liver tissue, pancreas tissue, pancreatic ductal tissue, spleen tissue, thymus tissue, Peyer's patch tissue, lymph nodes tissue, thyroid tissue, epidermis tissue, dermis tissue, subcutaneous tissue, heart tissue, lung tissue, vascular tissue, endothelial tissue, blood cells, bladder tissue, kidney tissue, digestive tract tissue, esophagus tissue, stomach tissue, small intestine tissue, large intestine tissue, adipose tissue, uterus tissue, eye tissue, lung tissue, testicular tissue, ovarian tissue, prostate tissue, connective tissue, endocrine tissue, and mesentery tissue.
  • tissues such as pancreatic tissue, liver tissue, smooth muscle tissue, striated muscle tissue, cardiac muscle tissue, bone tissue, bone marrow tissue, bone
  • the cells that are employed in the methods of the present invention are hematopoietic stem cells.
  • the hematopoietic stem cells can be obtained, for example, from the blood or bone marrow of a subject. Further, stem cells of different tissue types (other than hematopoitic stem cells) can be obtained from the blood.
  • the stem cells to be expanded can be isolated from any organ of any mammalian organism, by any means known to one of skill in the art.
  • the stem cells can be derived from embryonic or adult tissue.
  • One of skill of the art can determine how to isolate the stem cells from the particular organ or tissue of interest, using methods known in the art.
  • the stem cells are isolated from same as prior paragraph.
  • the stem cells can be obtained from blood or bone marrow.
  • IMDM Iscove's modified Dulbecco's Media
  • DMEM DMEM
  • KO-DMEM DMEM/F12
  • RPMI 1640 McCoy's 5 A medium
  • minimum essential medium alpha medium .alpha.-MEM
  • F-12K nutrient mixture medium Kaighn's modification, F-12K
  • X- vivo 20 Stemline, CClOO, H2000, Stemspan, MCDB 131 Medium, Basal Media Eagle (BME), Glasgow Minimum Essential Media, Modified Eagle Medium (MEM), Opti-MEM I Reduced Serum Media, Waymouth's MB 752/1 Media, Williams Media E, Medium NCTC- 109, neuroplasma medium, BGJb Medium, Brinster's BMOC-3 Medium, CMRL Medium, CO 2 -Independent Medium, Leibovit
  • IMDM Iscove's modified Dulbecco's Media
  • DMEM DMEM
  • KO-DMEM DM
  • growth factors can be added.
  • growth factors and other components include, but are not limited to, thrombopoietin (TPO), stem cell factor (SCF), IL-I, IL-3, IL-7, flt-3 ligand (fit-3L), G-CSF, GM-CSF, Epo, FGF-I, FGF-2, FGF-4, FGF-20, IGF, EGF, NGF, LIF, PDGF, bone morphogenic proteins (BMP), activin-A, VEGF, forskolin, glucocorticords, and the like.
  • the media can contain either serum such as fetal calf, horse, or human serum, or more preferably, serum substitution components.
  • serum substitutes have included bovine serum albumin (BSA), insulin, 2-mercaptoethanol and transferrin (TF).
  • BSA bovine serum albumin
  • TF transferrin
  • the stem cells can then be stored for a desired period of time, if needed.
  • Stem cell storage methods are well-known to those of skill in the art.
  • the stem cells can be sorted prior to administration by methods known in the art, using, for example, antibody technology such as fluorescence activated cell sorting (FACS), magnet activated cell sorting methods (e.g., magnetic resonance beads), column chromatography, or to isolate cells having the desired stem cell markers, or to remove unwanted, contaminating cell types having unwanted cell markers.
  • FACS fluorescence activated cell sorting
  • magnet activated cell sorting methods e.g., magnetic resonance beads
  • column chromatography or to isolate cells having the desired stem cell markers, or to remove unwanted, contaminating cell types having unwanted cell markers.
  • stem cells expressing a truncated recombinant GPCR amino acid sequence can be isolated from cells that do not expression a truncated recombinant GPCR amino acid sequence using any of these techniques.
  • the stem cells can have transferred an expression construct encoding a reporter comprising a GPCR or truncated recombinant GPCR prior to introduction of the cells to the subject at any stage in the preparation.
  • the GPCR may be a somatostatin receptor, SSTR2A, a truncated somatostatin receptor, a truncated SSTR2A, SSTR2delta314.
  • the reporter encodes a protein tag fused to the N- terminal end or C-terminal end of the truncated GPCR amino acid sequence.
  • the protein tag may or may not have enzymatic activity.
  • the protein tag may be, for example, hemagglutinin A, beta-galactosidase, thymidine kinase, transferrin, myc-tag, VP 16, (His) 6 -tag, FLAG, or chloramphenicol acetyl transferase.
  • GPCRs Recombinant G-Protein Coupled Receptors
  • GPCRs are a class of proteins involved in signal transduction, and are one of the largest receptor superfamilies known. These receptors are biologically important, and malfunction of these receptors has been shown to result in diseases such as Alzheimer disease, Parkinson disease, diabetes, dwarfism, color blindness, retinitis pigmentosa and asthma. GPCRs are also involved in depression, schizophrenia, sleeplessness, hypertension, anxiety, stress, renal failure and in several other cardiovascular, metabolic, neural, oncologic and immune disorders (Horn and Vriend, 1998). They have also been shown to play a role in HIV infection (Feng et al, 1996).
  • GPCRs have been characterized as having seven putative transmembrane domains that are connected by loops. The N-terminus is always extracellular and C-terminus is intracellular. The signal, such as an endogenous ligand or chemical moiety, is received at the extracellular N-terminus side. This signal is then transduced through the membrane to the cytosolic side where a heterotrimeric protein G-protein is activated which in turn elicits a response (see Horn and Vriend, 1998). GPCRs include a wide range of biologically active receptors, such as hormone receptors and neuronal receptors. Examples include, but are not limited to somatostatin receptors and adrenergic receptors..
  • aspects of the invention include non-invasive 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 a recombinant GPCR amino acid sequences.
  • the recombinant GPCR for example, may be a recombinant GPCR.
  • 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 Pub. No. 2002/0173626, which is herein specifically incorporated by reference in its entirety. Additional information regarding sequences of somatostatin receptors is set forth in Table 1 :
  • GPCR Accession Number pertains to nucleic acid sequence and encoded protein. Detailed information regarding the splice variants of human SSTR2 and its genomic structure can be found in Petersenn et al., 1999, herein specifically incorporated by reference. Additional information regarding the sequence of SSTR2 can be found in Yamada et al. (1992) and Vanetti et al. (1992).
  • 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 also includes nucleic acids encoding truncated GPCR polypeptide equivalents or other reporter polypeptide equivalents. 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 (He): 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,
  • the GPCR encoding sequences and other reporter sequences set forth herein 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.
  • the truncated recombinant GPCR amino acid sequence is a truncated recombinant somatostatin receptor amino acid sequence.
  • SSTR somatostatin receptors
  • 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 analogs, approved for lung imaging, are used in clinical practice to detect tumors over-expressing somatostatin receptors, such as neuroendocrine tumors.
  • the normal biodistribution and dosimetry of radiolabeled somatostatin analogs used for imaging clinically has been well studied.
  • the radiopharmaceutical is normally found in the kidneys, bladder, liver, spleen and bowel after intravenous injection. At the tracer doses used for imaging, no side-effects greater than placebo are found and patients are routinely imaged serially. Clinically, increased SSTR2 expression renders even small tumors detectable. PET based agents are also being developed.
  • 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. No functional (phenotypic changes) cellular changes were assessed such as effects on proliferation.
  • 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.
  • somatostatin can regulate secretion.
  • 32D hematopoietic cells cAMP appears to be required for SSTR2 mediated chemotaxis. The cytoplasmic C-terminus of the somatostatin receptor is involved in regulating cAMP.
  • HEK 293 human embryonic kidney
  • CHO Kl Chinese hamster ovary
  • 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
  • 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.
  • nNOS neuronal nitric oxide synthase
  • guanylate cyclase both of which appear necessary for SSTR2 mediated inhibition of proliferation in CHO cells and mouse pancreatic acinar cells.
  • the inhibition may also involve other phosphotyrosine phosphatases 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.
  • Truncation can be at either the N-terminus or the C-terminus or both termini.
  • aspects of the invention include transfecting a cell with an expression construct comprising a first region that is a nucleic acid sequence encoding a first reporter comprising a truncated recombinant GPCR amino acid sequence operatively linked to a first promoter sequence.
  • the GPCR may be a recombinant GPCR that produces or binds to or enzymatically acts upon agents that produce a detectable signal.
  • expression construct may include one or more additional nucleic acid sequences, such as additional reporters, additional coding regions, or additional promoters
  • 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.
  • a nucleic acid may encompass a double-stranded molecule or a triple-stranded molecule that comprises one or more complementary strand(s) or "complement(s)" of a particular sequence comprising a molecule
  • 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 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.
  • 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.
  • the expression vectors may contain nucleic acid sequences that serve other functions as well. 1. Promoters
  • 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.
  • 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.
  • 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 ah, 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 ah, 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.
  • operatively positioned means 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. Patents 4,683,202 and 5,928,906, each incorporated herein by reference).
  • Such promoters may be used to drive reporter expression, which include, but are not limited to GPCRs, ⁇ -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- selective, inducible, lineage-specific, or function-specific and/or useful under the appropriate conditions to direct expression of the introduced DNA segment, such as is advantageous in the production of proteins, recombinant proteins and/or peptides.
  • the promoter may be heterologous or endogenous or a combination thereof. The position of the promoter/ may be varied.
  • a first, second, third or fourth promoter of an expression cassette may be a constitutive, tissue selective, lineage specific, or function-specific promoter sequence that drives expression of a gene of interest, such as a reporter, a signaling sequence, a trafficking sequence, or a therapeutic gene.
  • 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-selective 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).
  • tissue-selective factor e.g., a tissue-selective factor
  • a “function-specific ppromoter sequence” is a control sequence that is a region of a nucleic acid sequence at which initiation and rate of transcription are controlled, wherein the sequence is active in cells and whose products perform a particular function of interest. Examples include insulin, T-cell receptor, immunoglobulin, hormone or paracrine promoters such as vascular endothelial growth factor, structural protein promoters such as dystrophin, intracellular components such as fat or melanin, or extracellular components such as cartilage.
  • Promoters useful in the practice of the present invention may be tissue-specific—that is, they are capable of driving transcription of a gene in one or a few normal or diseased tissue(s) while remaining largely “silent” or expressed at relatively low levels in other tissue types. It will be understood, however, that tissue-specific or tissue-selective promoters may have a detectable amount of "background” or “base” activity in those tissues where they are silent. The degree to which a promoter is selectively activated in a target tissue can be expressed as a selectivity ratio (activity in a target tissue/activity in a control tissue).
  • a tissue specific promoter useful in the practice of the present invention typically has a selectivity ratio of greater than about 1 :1.01, 1 :1.1, 1 :1.5, 1 :2, 1 :3, 1 :4, 1 :5 or more. Preferably, the selectivity ratio is greater than about 1 :1.5.
  • the promoter may also function in a reverse manner with decreased activity in the normal or diseased tissue(s) of interest. It will be further understood that certain promoters, while not restricted in activity to a single tissue type, may nevertheless show selectivity in that they may be active in one group of tissues, and less active or silent in another group. Such promoters are also termed "tissue specific" or "tissue selective,” and are contemplated for use with the present invention. For example, promoters that are active in a particular type of tissue may be therapeutically useful in diseases affecting the tissue that may be amenable to stem cell therapy.
  • the level of expression of a coding region under the control of a particular promoter can be modulated by manipulating the promoter region.
  • different domains within a promoter region may possess different gene-regulatory activities.
  • the roles of these different regions are typically assessed using vector constructs having different variants of the promoter with specific regions deleted (i.e., deletion analysis) or base pair(s) mutated.
  • Vectors used for such experiments typically contain a reporter sequence, which is used to determine the activity of each promoter variant under different conditions.
  • Application of such a deletion analysis enables the identification of promoter sequences containing desirable activities and thus identifying a particular promoter domain, including core promoter elements, those elements when deleted detrimentally effect characteristics of the promoter, such as but not limited to selectivity or transcription factor binding.
  • This approach may be used to identify, for example, the smallest region capable of conferring tissue specificity, or the smallest region conferring a robust transcriptional response when combined with other promoter elements, such as but not limited to the core CMV promoter or a mini-CMV.
  • promoters may be particularly advantageous in practicing the present invention.
  • 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.
  • promoters/enhancers include, but is not limited to Immunoglobulin Heavy Chain (Banerji et al, 1983; Gilles 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 Internal Ribosome Entry Sites
  • IRES elements are used to create multigene, or polycistronic, messages. IRES elements are able to bypass the ribosome scanning model of 5' methylated Cap dependent translation and begin translation at internal sites (Pelletier and Sonenberg, 1988). IRES elements from two members of the picornavirus family (polio and encephalomyocarditis) have been described (Pelletier and Sonenberg, 1988), as well an IRES from a mammalian message (Macejak and Sarnow, 1991) and further sequences as well as modified versions are envisioned in this application for invention. IRES elements can be linked to heterologous open reading frames.
  • 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.
  • 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 ah, 1997).
  • 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.
  • 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 may comprise a signal for the cleavage of the RNA, and it is more specific 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
  • aspects of the invention include transferring into a cell an expression construct comprising a nucleic acid sequence encoding a truncated recombinant GPCR operatively coupled to a promoter sequence.
  • Techniques pertaining to the transfer of expression constructs into cells are well-known to those of ordinary skill in the art. Exemplary techniques are discussed below.
  • transfer of an expression construct into a cell is accomplished using a viral vector.
  • 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.
  • the viral vector is a lentivirus vector. Lentivirus vectors have been successfully used in infecting stem cells and providing long term expression.
  • 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 al, 1992).
  • 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.
  • 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 mid- sized 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 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).
  • LTR long terminal repeat
  • 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.
  • 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. In addition, the availability of different viral control sequences with varying performance (temporal, strength, etc) makes it possible to control expression to a greater extent than in other systems. It also is an advantage that the virus has relatively few spliced messages, further easing genetic manipulations. HSV also is relatively easy to manipulate and can be grown to high titers. Thus, delivery is less of a problem, both in terms of volumes needed to attain sufficient MOI and in a lessened need for repeat dosings. For a review of HSV as a gene therapy vector, see
  • 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 it 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.
  • 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), nucleofection (Trompeter et al, 2003), 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 (Boussif 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).
  • the expression construct comprises a coding region that encodes a reporter other than a truncated recombinant GPCR.
  • 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.
  • reporters that can be imaged non-invasively or with non-invasive techniques are envisioned.
  • 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.
  • 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 is 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
  • 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.
  • the reporter sequence encodes a protein that can be visualized non- invasively such as the SSTR2 ⁇ 314.
  • 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.
  • F. Detectable Moieties In certain embodiments of the invention, a reporter, such as a truncated recombinant
  • GPCR amino acid sequence may be imaged by detecting its association with a detectable moiety.
  • a "detectable moiety” is defined herein to refer to any molecule that can attach, either directly or indirectly, to a reporter. Examples of detectable moieties are set forth above.
  • the detectable moiety is a ligand.
  • a ligand is defined herein to refer to an ion, a peptide, a oligonucleotide, aptamer, a molecule, a small 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 in these embodiments, may bind to or associate with the reporter.
  • 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 detectable moiety 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 l u 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, an ultrasound agent, an optical agent, or an MRI contrast agent.
  • a detectable moiety 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 and can be employed as detectable moieties. Examples include, but are not limited to 67 Ga, 68 Ga, "" 1 Tc, 111 In, 123 I, 125 I, 131 I, 169 Yb, 60 Cu, 61 Cu, 64 Cu, 62 Cu, 201 Tl, 72 A, and 157 Gd.
  • 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.
  • 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 of a detectable moiety 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).
  • Some aspects of the present invention pertain to methods for tracking the location of a cell in a subject that involve detecting the location of the cell in the subject by contacting the cell with a detectably moiety that binds to the truncated recombinant GPCR that is expressed in the cell.
  • Detection of the expressed GPCR 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 detctable moiety to a subject, wherein the detectable moiety is directed to the reporter amino acid sequence.
  • the detectable moiety is a radiolabeled probe, such as l u In-octreotide.
  • the detectable moiety is a probe that can be imaged optically, such as by fluorescence, near infrared, infrared, MR, or ultrasound. Any method known to those of ordinary skill in the art for measuring a signal derived from a reporter or an associated detectable moiety that attaches to the reporter is contemplated for inclusion in the present invention. Exemplary methods of detecting are as follows.
  • gamma camera imaging is contemplated as a method of imaging that can be utilized for measuring a signal derived from the reporter.
  • 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.
  • Single photon emission tomography (SPECT) may also be performed for three dimensional localization. 2.
  • CT Computerized Tomography
  • 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.
  • 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. Examples of these agents include iothalamate, iohexol, diatrizoate, iopamidol, ethiodol, and iopanoate. Gadolinium agents have also been reported to be of use as a CT contrast agent (see, e.g.,
  • 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
  • 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.
  • PET and SPECT Imaging modalities that provide information pertaining to information at the cellular level, such as cellular viability, include positron emission tomography (PET) and single- photon emission computed tomography (SPECT).
  • PET positron emission tomography
  • SPECT single- photon emission computed tomography
  • SPECT single-photon emission computed tomography
  • 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.
  • 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 of the eyes.
  • 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 erythrosin, 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.
  • Optical imaging includes near infrared imaging and infrared imaging. Near infrared imaging has more tissue penetration and less background. 6.
  • Ultrasound imaging has been used 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.
  • imaging using more than one modality is performed.
  • the imaging modality may include, but are not limited to, CT, MRI, PET, SPECT, ultrasound, or optical imaging.
  • Other examples of imaging modalities known to those of ordinary skill in the art are contemplated by the present invention.
  • the imaging modalities are performed at any time during or after administration of the composition comprising the diagnostically effective amount of the compound that comprises two imaging moieties.
  • 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. 8. Imaging of a Subject Following Stem Cell Administration
  • Imaging of a cell and/or its progeny can be performed following introduction of a cell into a subject. For example, imaging can be performed after about 1 second, 1 minute, 1 hour, 1 day, 1 week, 1 month, 1 year, or any longer period of time following administration of the cell. In some embodiments, imaging and biodistribution analysis can be performed as described by Yang et ah, 2005. In other embodiments, imaging may be preformed after approximately one and one-half weeks.
  • One of ordinary skill in the art would be familiar with generating a protocol to imaging cells, such as stem cells, following introduction of cells into a subject.
  • Imaging of a cell and/or its progeny that include an expressed truncated recombinant GPCR can be performed for several purposes. For example, imaging can be performed to follow the transit of cells, such as stem cells, in the body following introduction of the cells into a subject. Imaging can also be used to assess cell viability following introduction of the cells into a subject, and over the course of time. Further, imaging can also be performed to assess stem cell or immune cell localization in a subject. For example, placing the reporter under the control of a constitutive promoter would provide for constant expression that may be used to assess localization and viability of the cell. Imaging can be used to assess trans/differentiation or fusion. For example, placing the reporter under the control of a tissue-selective promoter sequence would provide for expression of a particular reporter only upon trans/differentiation or fusion of a cell or its progeny to a particular tissue/cell type.
  • imaging can be performed to assess an immune cell, stem cell or its progeny's expression from a promoter of a gene whose product performs a function of interest following introduction of the cell into a subject.
  • placing a reporter in the expression construct under the control of a function-specific promoter would provide for expression of the reporter in stem cells until trans/differentiation or fusion.
  • expression may occur upon differentiation of the cell into a cell capable of performing a specific function.
  • T-cell activation may be assessed using promoter elements that initiate transcription upon T-cell activation.
  • the reporter may be linked to a gene of interest, for example by an IRES or a bidirectional promoter, so that expression of the reporter may be used to track not only its own expression, but also that of the gene of interest.
  • genes of interest include those whose products may function in homing, implantation or differentiation.
  • multiple promoter-reporter constructs transferred into a cell combinations of the above may be evaluated including in vivo.
  • viability, localization, differentiation, functional expression, and indirect evaluation of expression of a linked gene of interest may be evaluated.
  • different promoters and different reporters that can be identified either simultaneously or serially may need to be employed.
  • Simultaneous evaluation may be performed for example if the reporter or its detectable moiety have separable characteristics, for example, different energies of emission of gamma rays that can be separated by a gamma camera.
  • a combination of more than one imaging technique can be used to determine trafficking, viability, and/or differentiation of the cells.
  • MR and ⁇ -camera imaging can be used to determine the biodistribution of radiopharmaceutical in tumors. Imaging can be performed following administration of a subject with a detectable moiety. Because the detectable moiety will have a limeited physical and biological life, imaging of the reporter can be performed repeatedly.
  • cells that have been exposed to a detectable moiety can be introduced into the subject, and then the subject subjected to one or more imaging techniques following introduction of the cells. Imaging can be performed a single time or more than one time point following introduction of the cells into the subject allowing serial evaluation. Image acquisition can be performed by any method known to those of ordinary skill in the art.
  • the reporter within the cells can be imaged both in vivo and ex vivo.
  • ex vivo imaging occurs on a biopsy sample of tissue obtained from the subject following introduction of the cell into the subject.
  • vzVo-imaging can also be performed using any of a variety of modalities known to those of ordinary skill in the art.
  • ex vivo evaluation occurs on a biopsy sample of tissue obtained from the subject following introduction of the cell into the subject.
  • Ex vivo evaluation for the reporter can be performed using a variety of techniques including but not limited to autoradiography, immunologic techniques such as immunohistochemistry, ELISA or Western blotting, PCRTM, optical, CT, MR, nuclear imaging, or ultrasound.
  • immunologic techniques such as immunohistochemistry, ELISA or Western blotting, PCRTM, optical, CT, MR, nuclear imaging, or ultrasound.
  • PCRTM optical, CT, MR, nuclear imaging, or ultrasound.
  • in vzVo-imaging can also be performed using any of a variety of modalities known to those of ordinary skill in the art.
  • the GPCR is used for the purpose of non-invasive or invasive imaging.
  • the GPCR is a somatostatin receptor.
  • the somatostatin receptor is SSTR2A.
  • the GPCR is truncated.
  • the GPCR is truncated and inhibited in eliciting signaling and/or phenotypic change and/or internalization.
  • the truncated GPCR is truncated a somatostatin receptor.
  • the truncated somatostatin receptor is truncated SSTR2A.
  • the truncated SSTR2A is SSTR2 ⁇ 314.
  • transgenic animals via embryo manipulation and microinjection, particularly animals such as mice, have become conventional in the art, e.g. (U.S. Pat. No. 4,870,009; U.S. Pat. No. 4,736,866; U.S. Pat. No. 4,873,191).
  • Other non-mice transgenic animals may be made by similar methods.
  • a transgenic founder animal which can be used to breed additional transgenic animals, can be identified based upon the presence of the transgene in its genome and/or expression of the transgene mRNA in tissues or cells of the animals.
  • Transgenic animals can be bred to other transgenic animals carrying other transgenes.
  • non-human animals is intended to include any vertebrate such as mammals, birds, reptiles, and amphibians. Suitable mammals include rodents ⁇ e.g., rat, mouse), non-human primates, sheep, dogs, cats, rabbits, and cows. Suitable birds include chickens, geese, and turkeys.
  • Transgenic animal refers to non-naturally occurring non- human animal in which one or more of the cells of the animal contain heterologous nucleic acids encoding human a truncated recombinant GPCR, that has been introduced by way of human intervention, such as by transgenic techniques well known in the art.
  • the nucleic acid is introduced into the cell, directly or indirectly by introduction into a precursor of the cell, by way of deliberate genetic manipulation, such as by microinjection or by infection with a recombinant virus.
  • This molecule may be integrated within a chromosome, or it may be extrachromosomally replicating DNA.
  • the animal is transgenic for truncated SSTR2 such as SSTR2A ⁇ 314.
  • the cells/tissues/organs from the transgenic animal may be studied in vitro or in vivo. This animal may be made to express the gene under the control of a constitutive, tissue specific, lineage specific, or functional promoter. Cells from the animal may be introduced into another animal not transgenic for the same reporter for studying cell trafficking, viability, engraftment, trans/differentiation or fusion, expression from a promoter of a gene of a function of interest, expression of a linked gene of interest, etc. as described elsewhere in the application.
  • stem cells or immune cells are extracted from the transgenic animal and studied as described above after injection into another animal with or without pathology.
  • immune cells are extracted from the transgenic animal and studied as described above after injection into another animal with or without pathology. The cells may be used after extraction or may be cultured and then introduced into another animal.
  • tissues such as islets and/or organs may be transplanted and the GPCR reporter may be used for example but not limited to track their viability, incorporation, and trafficking of cells that may migrate.
  • tissues such as islets and organs may be transplanted and the GPCR reporter may be used for example to track their viability, incorporation, and trafficking of cells that may migrate from the graft.
  • stem cells expressing the GPCR reporter may be made to differentiate into tissues or organs ⁇ e.g., embryoninc stem cells into heart) in vitro or in vivo and then be transplanted into another animal and the GPCR reporter may be used for example to track viability, incorporation and trafficking of cells that may migrate from the graft.
  • the invention includes a method of generating stem cells by obtaining cells, stem cells or immune cells from a transgenic animal.
  • bone marrow may be used as a stem cell source and be directly introduced into another animal.
  • Stem cells may be introduced into a subject with other cells in order to improve engraftment.
  • obtaining stem cells involves fractionating the cells into a fraction enriched with a stem cell and culturing the stem cells in a culture medium containing one or more growth factors. By this process, the stem cells will undergo mitotic expansion.
  • the invention contemplates the establishment and maintenance of cultures of stem cells as well as mixed cultures comprising stem cells, mature cells and mature cell lines.
  • stem cell cultures involve techniques that are well-known to those of ordinary skill in the art.
  • the cells of the invention may be maintained or stored in "cell banks" comprising either continuous in vitro cultures of cells requiring regular transfer, or, preferably, cells which have been cryopreserved.
  • cryopreservation of cells of the invention may be carried out according to methods known to those of ordinary skill in the art.
  • the cryopreserved cells of the invention constitute a bank of cells, portions of which can be "withdrawn” by thawing and then used to produce new stem cells, etc. as needed.
  • the cells of the invention may be used as ubiquitous donor cells, i.e., to produce new tissue for use in any subject (heterologous). J. Clinical Applications and Pharmaceutical Preparations
  • Treatment refers to administration or application of a drug or therapy
  • therapeutic benefit refers to anything that promotes or enhances the well-being of the subject with respect to the medical treatment of his condition. This includes, but is not limited to, a reduction in the frequency or severity of the signs or symptoms of a disease.
  • a “disease” or “health-related condition” can be any pathological condition of a body part, an organ, or a system resulting from any cause, such as infection, genetic defect, trauma, and/or environmental stress.
  • the cause may or may not be known. Examples of such conditions include cancer and diabetes.
  • Prevention and “preventing” are used according to their ordinary and plain meaning to mean “acting before” or such an act.
  • those terms refer to administration or application of an agent, drug, or remedy to a subject or performance of a procedure or modality on a subject for the purpose of blocking the onset of a disease or health-related condition.
  • the cells of the invention may be applied to treat subjects requiring the repair or replacement of body tissues resulting from disease or trauma. Treatment may entail the use of the cells of the invention to produce or induce new tissue, and the use of the tissue thus produced, according to any method presently known in the art or to be developed in the future. For example, the cells of the invention may be given systemically, implanted, injected or otherwise administered directly to the site of tissue damage so that they will produce or induce new tissue in vivo.
  • stem cells the mature cells produced from these stem cells, and the cell lines derived from these stem cells can be used: (1) to screen for the efficacy and/or cytotoxicity of compounds, allergens, growth/regulatory factors, pharmaceutical compounds, etc.; (2) to elucidate the mechanism of certain diseases; (3) to study the mechanism by which drugs operate; (4) to diagnose, monitor and treat cancer in a patient; (5) for gene therapy; and (6) to produce biologically active products, to name but a few uses.
  • immune cells can be applied in methods of therapy. These cells may be active against cells expressing a particular antigen. Methods of therapy involving immune cells involve techniques well-known to those of ordinary skill in the art.
  • compositions of the present invention involve introducing a pharmaceutically acceptable dose of cells encoding a truncated recombinant GPCR.
  • Pharmaceutical compositions of the present invention comprise a therapeutically or diagnostically effective amount of the cells of the present invention.
  • pharmaceutically acceptable or “therapeutically effective” or “diagnostically effective” refers to compositions of cells of the present invention that do not produce an unacceptable adverse, allergic or other untoward reaction when administered to a subject, such as, for example, a human or a laboratory animal ⁇ e.g., mouse, rat, dog), as appropriate.
  • compositions comprising a therapeutically effective amount or "a composition comprising a diagnostically effective amount” includes any and all solvents, dispersion media, antioxidants, preservatives ⁇ e.g., antibacterial agents, antifungal agents), cell culture media, isotonic agents, salts, preservatives, drugs, drug stabilizers, gels, and combinations thereof, as would be known to one of ordinary skill in the art.
  • the cells of the present invention can be introduced to a subject by any method known to those of ordinary skill in the art. Examples include intravenously, intradermally, intraarterially, intraperitoneally, intralesionally, intracranially, intraarticular Iy, 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, directly into a heart chamber, directly injected into the organ or portion of organ or diseased site of interest, or by other method or any combination of the forgoing as would be known to one of ordinary skill in the art.
  • compositions of the present invention administered to a subject 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.
  • the present invention contemplates methods of preventing, inhibiting, or treating such diseases or conditions in a subject by administration of a cell that has been transfected with an expression construct encoding a truncated recombinant GPCR amino acid sequence operatively linked to a promoter. Aspects of the invention also include the use of the methods and compositions of the invention in combination with other therapies, as discussed in greater detail below.
  • 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 cell as described herein.
  • the disease may be a disease amenable to stem cell therapy. Examples include cancer, diabetes, cardiovascular disease, neurological disease, neurodegenerative disease, genetic disease, liver disease, infection, trauma, toxicity, or immunological disease. Additional diseases are discussed elsewhere in this specification.
  • the disease may be a hyperproliferative disease.
  • a hyperproliferative disease is a disease associated with the abnormal growth or multiplication of cells.
  • the hyperproliferative disease may be a disease that manifests as lesions in a subject. Exemplary hyperproliferative lesions include pre-malignant lesions, cancer, and tumors.
  • the cancer can be any type of cancer including those derived from mesoderm, endoderm, or ectoderm such as blood, heart, lung, esophagus, muscle, intestine, breast, prostate, stomach, bladder, liver, spleen, pancreas, kidney, neurons, myocytes, leukocytes, immortalized cells, neoplastic cells, tumor cells, cancer cells, duodenum, jejunum, ileum, cecum, colon, rectum, salivary glands, gall bladder, urinary bladder, trachea, larynx, pharynx, aorta, arteries, capillaries, veins, thymus, lymph nodes, bone marrow, pituitary gland, thyroid gland, parathyroid glands, adrenal glands, brain, cerebrum, cerebellum, medulla, pons, spinal cord, nerves, skeletal muscle, smooth muscle, bone, testes, epidiymides, prostate, seminal vesicles
  • 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.
  • the present invention include treating a subject with a disease in combination with the methods for tracking the location of a cell set forth herein.
  • the invention pertains to a method of tracking the location of a cell in a subject and treating a subject with a disease.
  • the disease can be any disease, but in particular embodiments the disease is one for which stem cell therapy is known or suspected to be of benefit.
  • SSTR2A and the deletion mutant were constructed from a phage (ATCC, Rockville, MD) containing the SSTR2A insert by PCR using the following oligonucleotide primers: SSTR2 FL-F (SEQ ID NO: 23) (TCC CCG CGG CAT GGA CAT GGC GGATGA), SSTR2 FL-R (SEQ ID NO: 24) (AAT CTG CAG
  • the reverse primers contained a Pst I restriction site and a stop codon.
  • the full-length SSTR2A and SSTR2 ⁇ 314 inserts were ligated into the Sac II and Pst I inserts in pDisplay vector (Invitrogen, Carlsbad, CA) separately. The inserts were placed downstream of the membrane localization sequence (5 '
  • IgK leader IgK leader
  • sequence for hemagglutinin A (HA) epitope tag The inserts were confirmed by sequencing.
  • HT 1080 human fibrosarcoma, ATCC, Rockville, MD
  • HEK293 human fibrosarcoma, ATCC, Rockville, MD
  • Adherent cultures were incubated at 37 C in a mixture of 5% CO 2 and 95% air.
  • 2 ⁇ g DNA mixed with 3 ⁇ l FuGen ⁇ (Invivogen, San Diego, CA) transfection reagent in 50 ⁇ l DMEM was added to 1 x 10 5 cells in 24-well plates per manufacturer's instructions.
  • lipofectin Invitrogen, Carlsbad, CA
  • Cells were selected with 1 mg/ml G418.
  • ELISA and immunofluorescence were performed as previously described (Kundra et al, 2002). Prospective colonies expressing fusion protein were screened by ELISA, and expression in selected clones was quantified using a quantitative ELISA. 25mU/ml horseradish peroxidase (HRP) conjugated rat anti-HA antibody (clone 3F10 from Roche, Indianapolis, IN) was used for ELISA. Indirect immunofluorescence employed an anti-HA primary antibody (HA-11, Babco, Richmond, CA).
  • HRP horseradish peroxidase
  • Indirect immunofluorescence employed an anti-HA primary antibody (HA-11, Babco, Richmond, CA).
  • PCR was performed with a thermal cycler (Eppendorf, Westbury, NY). The conditions were one cycle each (30 min at 50 C and 2 min at 94 C) for cDNA synthesis and denaturation, 35 cycles (15 s at 94°C, 30 s at 60°C, and 1 min at 72°C) for PCR amplification, and one cycle (5 min at 72°C) for final extension. PCR reactions were carried out in a total volume of 50 ⁇ l containing 0.2 ⁇ M primers and 2 units of taq in EcoTaq buffer.
  • SSTR2-FL-F (SEQ ID NO:26) ATGGACATGGCGGATGAGCCACTCAATGG with SSTR2-FL-R: (SEQ ID NO:27) TCAGATACTGGTTTGGAGGTCTCCATTGAG for the wild-type receptor transcript and SSTR2-F1-F: (SEQ ID NO:28) ATGGACATGGCGGATGAGCCACTCAATGG with SSTR2- ⁇ 314-R: (SEQ ID NO:29) AGAAGGCATATAGGATAGGGTTGGACAGC for the portion of the transcript coding up to amino acid 314.
  • Binding assay Cells were harvested in binding buffer (50 mM Tris-HCl, pH 7.8, containing 1 mM EGTA, 5 mM MgCl 2 , 10 ⁇ g/ml leupeptin, 10 ⁇ g/ml pepstatin, 200 ⁇ g/ml bacitracin, and 0.5 ⁇ g/ml aprotinin) and were centrifuged at 12,000 rpm for 10 min at 4 C. The pellet was homogenized with a polytron at 50 Hz for 20 s in binding buffer. Five micrograms per well of the membrane preparation was used for the radioligand binding studies (Birzin et al, 2002).
  • Each well of the GF/B multi-screen plates (Millipore, Bedford, MA) was pretreated with 100 ⁇ l of 0.1% polyethyleneimine per well for 2 h.
  • the solution was removed using a multiscreen vacuum manifold (Millipore, Bedford, MA) and then the filter plates were washed one time with 200 ⁇ l of 50 mM Tris-HCI (pH 7.8).
  • Assay reagents were added to the washed plates in the following order: 160 ⁇ l membrane and 40 ⁇ l (200 nM-0.7 nM final concentration) 111 In-octreotide (Mallinckrodt, St.
  • cAMP or cGMP levels Intracellular levels of cAMP or cGMP in response to somatostatin were measured in triplicate by modified ELISA methods.
  • the cells were incubated at 37 C for 30 min in 1 ml PBS with or without forskolin (10 7 M), or somatostatin (10 ⁇ n - 10 "7 M) plus forskolin (10 7 M).
  • somatostatin 10 ⁇ n - 10 "7 M
  • the cells were incubated with or without somatostatin (10 11 - 10 "7 M).
  • the levels of cAMP or cGMP in the cell lysates were detected using commercially available ELISA kits (Sigma-Aldrich, St. Louis, MO). The concentrations of cAMP or cGMP were calculated from standard curves.
  • mice were injected subcutaneous Iy. Each mouse received three inoculations: left thigh, HT1080 cells transfected with vector; left shoulder, HT1080 cells expressing wild-type fusion protein; and right shoulder, HT 1080 cells expressing ⁇ 314 fusion protein. After approximately one and one-half weeks, imaging and bio distribution analysis was performed as described (Yang et al, 2005). The mice were injected intravenously via the tail vein with 300 ⁇ Ci of lu In-octreotide (Mallinckrodt, St. Louis, Mo).
  • mice were imaged with a 4.7 T small animal MR (Bruker, Billerica, MA) using a T2- weighted fast spin echo sequence. Twenty-four hours after radiopharmaceutical injection, the animals were imaged for 10 min using a ⁇ -camera fitted with a medium-energy collimator (mCAM, Siemens Medical Solutions, Hoffman Estates, IL). Acquisition was with a 512 x 512 matrix that was compressed to a 256 x 256 matrix for viewing and measurement. A combination of the in vivo MR and ⁇ -camera imaging was used to determine the biodistribution of the radiopharmaceutical in tumors.
  • mCAM medium-energy collimator
  • HEK 293 and human fibrosarcoma (HT 1080) cells do not endogenously express somatostatin receptors.
  • FIG. IA no SSTR subtype expression was found using RT-PCR of RNA extracts from either cell line. RT-PCR of the same RNA extracts did result in a product for the ubiquitiously expressed ⁇ -actin, confirming the quality of the RNA. Functionality of the SSTR subtype primers was confirmed by PCR of SSTR subtype cDNA.
  • HEK 293 human fibrosarcoma
  • HT 1080 human fibrosarcoma
  • RT-PCR Reverse transcriptase polymerase chain reaction
  • Primers for either wild type receptor or the ⁇ 314 mutation resulted in appropriately sized products when RNA from either HEK293 or HT 1080 cells transfected with wild type receptor was used.
  • only primers for ⁇ 314 resulted in appropriately sized products when RNA from HEK293 or HT 1080 cells transfected with the ⁇ 314 receptor was used; whereas, no product was seen when primers for the full length receptor were used.
  • HEK293 or HT1080 clones transfected with wild-type or ⁇ 314 SSTR2 gene chimeras express the predicted RNA transcripts.
  • Immunofluorescence demonstrated expression of wild-type or ⁇ 314 SSTR2 fusion protein on the cell membranes of transfected HT 1080 or HEK293 clones, and background signal on vector transfected cells.
  • ELISA enzyme linked immunosorbent assay
  • forskolin was utilized to activate adenylate cyclase.
  • Addition of forskolin to HEK293 or HT 1080 cells resulted in cAMP production whether the cells expressed wild type or ⁇ 314 fusion protein.
  • Addition of somatostatin inhibited forskolin- induced production of cAMP in either cell line expressing full length receptors.
  • somatostatin did not inhibit forskolin-induced production of cAMP in HEK293 or HT 1080 cells expressing the ⁇ 314 mutant. Findings in two different human cell types imply that the human HA-SSTR2 ⁇ 314 mutant is deficient in regulating the cAMP signaling pathway.
  • ⁇ 314 mutant Cellular activity, mitosis. Function of the ⁇ 314 mutant was further assessed at the cellular level. Whether deleting amino acids distal to 314 affects the capacity of the SSTR2A to inhibit proliferation was tested in HEK293 and HT 1080 cells, as shown in FIG. 5 A, 5B, 5C, and 5D. Somatostatin is not stable in culture media long term, therefore, a stabilized analogue, Sandostatin, was used to maintain receptor activation. Serum stimulation promoted growth in HEK293 cells or HT 1080 cell clones. As predicted, Sandostatin inhibited serum- induced proliferation in HEK293 cells or HT 1080 cells expressing full length receptors. In contrast, HEK293 or HT 1080 cells expressing the ⁇ 314 mutants were muted in inhibiting serum-induced proliferation. Thus, the ⁇ 314 SSTR2 mutant is deficient in inhibiting cellular proliferation.
  • Radiopharmaceutical uptake by tumors was quantified by in vivo biodistribution analysis (O'Carroll et ah, 1993). To control for tumor weight, prior to gamma camera imaging, the animals underwent T2 -weighted fast spin echo (FSE) magnetic resonance (MR) imaging to obtain the weight of each tumor.
  • FSE fast spin echo
  • MR magnetic resonance
  • the biodistribution analysis confirmed the imaging findings that the SSTR2 ⁇ 314 receptor is amenable to imaging in vivo.
  • Planar ⁇ -camera images of a nude mouse demonstrated that tumors derived from HT1080 cells expressing HA-wt SSTR2 or expressing HA-SSTR2 ⁇ 314 were visible. In contrast, the tumor derived from HT 1080 cells transfected with vector was not visible.
  • HS-5 human bone marrow messencymal cells, ATCC, Rockville, MD
  • ⁇ -MEM fetal bovine serum
  • Ix penicillin- streptomycin mixture 10% fetal bovine serum
  • Adherent cultures were incubated at 37 C in a mixture of 5% CO 2 and 95% air. Gene transfer was performed using nucleofection.
  • Binding assay The binding assay was as described in Example 1 above except a single dose (10 "7 M) of l u In-octreotide was used. Competition was performed using 10 6 M somatostatin. Growth Inhbition Assay. The growth inhibition assay was as described in Example 1 except a single dose (10 "7 M) of l u In-octreotide was used.
  • Osteogenic induction Cells were induced using osteogenic induction medium using the manufacturers instructions (Cambrex, East Rutherford, NJ) and calcium production was evaluated using the StanbioTotal Calcium LiquiColor using the manufacturers instructions (Stanbio Laboratory, Boerne, TX).
  • Adipogenic induction Cells were induced using the adipogenic induction medium using the manufacturers instructions (Cambrex, East Rutherford, NJ). After induction, the cells were rinsed in phosphate buffered saline (PBS), fixed in 10% buffered formalin and stained with Oil Red O to visualize lipid vacuoles.
  • PBS phosphate buffered saline
  • results of a quantitative ELISA using an antibody to the HA-domain showed that stably transfected HS-5 cells express similar amounts of HA-SSTR2 ⁇ 314 (A314) or HA- wtSSTR2 (wt) (FIG. 7). Further, it was found that stably transfected HS-5 cells expressing A314 or HA-wt SSTR2 (wt) bind similar amounts of 111 In octreotide (FIG. 8).
  • HA-SSTR2 ⁇ 314 is signaling deficient for the cAMP pathway in human bone marrow mesenchymal cells, HS-5 (FIG. 9).
  • HA-wt SSTR2 decreased forskolin-induced cAMP production (wt) whereas HA- SSTR2 ⁇ 314 (A314) did not when expressed in HS-5 cells.
  • HA-SSTR2 ⁇ 314 was found to be signaling deficient for the cGMP pathway in human bone marrow mesenchymal cells, HS-5 (FIG. 10).
  • HA-wt SSTR2 (wt) incited cGMP production, whereas HA-SSTR2 ⁇ 314 ( ⁇ 314) does not when expressed in HS-5 cells.
  • HA-SSTR2 ⁇ 314 was found to be deficient in inhibiting cell growth in human bone marrow mesenchymal cells, HS-5 (FIG. 11).
  • HS-5 human bone marrow mesenchymal cells
  • HA-wt SSTR2 wt
  • HA-SSTR2 ⁇ 314 A314 did not when expressed in HS-5 cells (P ⁇ 0.05).
  • HA-SSTR2 ⁇ 314 ⁇ 3114
  • HA-wt SSTR2 wt
  • calcium phosphate production was equivalent in cells transfected with vector or HA-SSTR2 ⁇ 314, but decreased in cells transfected with HA-wt SSTR2 (FIG. 12).
  • human bone marrow mesenchymal cells, HS-5, expressing HA-SSTR2 ⁇ 314 differentiate into adipocyte lineage.
  • HS-5 expressing HA-SSTR2 ⁇ 3144
  • FIG. 14 HS-5 cells stably transfected with HA-SSTR2 ⁇ 314, HA-wt SSTR2, or vector were exposed to 10 ⁇ 7 M lu In-octreotide for two hours, washed and then injected into C57/B16 mice via tail vein.
  • a control that did not include cells was incubated with 10 ⁇ 7 M lu In-octreotide for two hours, washed and then injected into C57/B16 mice via tail vein.
  • FIGS. 15-20 demonstrate that cells expressing the SSTR2 ⁇ 314 without a tag behave similarly to those with the tag.
  • a tag can be important, for example, for differentiating endogenous from exogenous SSTR2 expression and for reducing costs of in vitro and in vivo assessment, but may not be desirable under some circumstances.
  • a SSTR2 ⁇ 314 reflecting only human-derived sequences may be advantageous, for example, to reduce the possibility of an immune reaction to a tag or to decrease the already small size of the insert (of the reporter gene) in a construct.
  • Human peripheral white blood cells Human peripheral white blood cells were isolated via a Ficoll gradient. Each 10 ml of blood collected in a heparinized tube was mixed with 25 ml of phosphate buffered saline without calcium or magnesium and layered upon 15 ml of Histopaque-1077 (Sigma- Aldrich, St. Louis, MO) in a 50 ml conical tube and centrifuged at 4°C at 400 x g for 20 minutes. The buffy coat was resuspended in PBS and centrifuged at 4°C at 400 x g for 20 minutes. The cells were then washed again with PBS and transferred to a flask containing ⁇ -MEM with 20% fetal calf serum.
  • Histopaque-1077 Sigma- Aldrich, St. Louis, MO
  • the cells were grown in a humidified 5% CO 2 incubator at 37°C. Some cells were transferred to a slide and stained with Hematoxylin.
  • the white blood cells were infected with 10 11 adenovirus particles containing an insert for HA-wt SSTR2, HA-SSTR2 ⁇ 314, or control. Expression was confirmed by ELISA. Imaging. 10 6 white blood cells infected with HA-SSTR2 ⁇ 314 HA-wt SSTR2, or vector were exposed to 10 "7 M 111 In octreotide for two hours, washed and then injected into nude mice via tail vein.
  • HA-SSTR2 ⁇ 314 expression of HA-SSTR2 ⁇ 314 by isolated human peripheral white blood cells. It was found that isolated human peripheral blood white blood cells infected with adenovirus containing an insert for HA-SSTR2 ⁇ 314 ( ⁇ 314) or HA-wt SSTR2 (wt) express the fusion proteins. Results of quantitative ELISA using an antibody to the HA-domain are shown in FIG. 22. Expression of HA-wt SSTR2 (wt) was greater than that of HA-SSTR2 ⁇ 314 ( ⁇ 314) in the sets of cells used for the cell trafficking experiments discussed below. In vivo imaging of cell trafficking of isolated human peripheral white blood cells expressing HA - SSTR2 ⁇ 314.
  • White blood cells were infected with adenovirus containing an insert for HA-wt SSTR2, HA-SSTR2 ⁇ 314, or control were exposed to 10 ⁇ 7 M 111 In- octreotide for two hours, washed and then injected into nude mice via tail vein. A no cell control was incubated with 10 ⁇ 7 M ul In-octreotide for two hours, washed and then injected into nude mice via tail vein. Increased signal was found to overlie the lungs in mice injected with white blood cells infected with adenovirus containing an insert for HA-SSTR2 ⁇ 314 or HA-wt SSTR2 (FIG. 23).
  • mice production A construct containing a ubiquitin promoter-intron-HA- SSTR2 ⁇ 314-bovine growth hormone polyA was introduced into B6D2F1 mouse oocytes using pronuclear injection. Mice were bred in order to obtain homozygotes.
  • Western blotting was performed using an overnight exposure at 4°C to a mouse anti HA-I l antibody (1 :2000 dilution, Covance, Berkley, CA). After washing, an HRP-conjugated secondary goat-antimouse IgG antibody (1 :4000 dilution, Biorad Laboratories, Hercules, CA) was used for detection.
  • FIG. 24 Results Exemplary constructs incorporating HA-SSTR2 ⁇ 314 are shown in FIG. 24.
  • FIG. 24 Results Exemplary constructs incorporating HA-SSTR2 ⁇ 314 are shown in FIG. 24.
  • FIG. 24A shows examples of constructs using constitutive promoters (viral-CMV, and human - ubiquitin).
  • FIG. 24B shows a construct incorporating a functional and tissue-selective promoter as a marker for hepatocytes, since albumin expression is essentially restricted to hepatocytes.
  • FIG. 24C shows an example of an amplified functional and tissue-selective promoter (miniCMV) for amplifying expression from the albumin promoter.
  • miniCMV amplified functional and tissue-selective promoter
  • Introns need not be used, but can be helpful to increase expression in transgenic animals, and their positions in the construct may be varied.
  • Transgenic mice were created using a construct with an ubiquitin promoter for driving expression of HA-SSTR2 ⁇ 314. Transgenic mice were found to express HA-SSTR2 ⁇ 314 mRNA in multiple organs (FIG. 21).
  • FIG. 25 demonstrates results of a reverse transcriptase- polymerase chain reaction of RNA derived from transgenic mice or non-transgenic parental strain mice. Primers for HA-SSTR2 ⁇ 314 demonstrated expression of HA-SSTR2 ⁇ 314 in only transgenic mice.
  • transgenic mice express HA-SSTR2 ⁇ 314 protein in multiple organs, including bone marrow.
  • Results of Western blotting of protein derived from transgenic mice or non-transgenic parental strain mouse demonstrated expression of HA- SSTR2 ⁇ 314 in only transgenic mice (FIG. 26).
  • FIG. 27 demonstrates specific expression of HA-SSTR2 ⁇ 314 protein in the liver of the transgenic mouse. No expression is seen in the wild type mouse.
  • FIG. 27 shows that tissue-specific expression can be obtained with the signaling deficient reporter, HA- SSTR2 ⁇ 314. This is important, for example, for evaluating context specific expression and differentiation such as of stem cells, and for creating transgenic mice that express the signaling deficient reporter in specific tissues.

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Abstract

L'invention concerne des procédés pour repérer l'emplacement d'une cellule et/ou de sa descendance, tel qu'une cellule souche, chez un sujet. Lesdits procédés impliquent (a) l'obtention d'une cellule ; (b) la transfection de la cellule avec une construction d'expression qui comporte une première zone de codage codant un premier rapporteur, lequel inclut une séquence d'acide aminé du récepteur (GPCR) de recombinaison tronqué associé à la protéine G à sept domaines transmembranaire, fonctionnellement couplée à une première séquence de promoteur ; (c) la présentation de la cellule au sujet ; et (d) la détection de l'emplacement de la cellule chez le sujet par la mise en contact de la cellule avec un groupe caractéristique détectable qui se lie à la GPCR recombinante tronquée ainsi que la réalisation de l'imagerie du groupe caractéristique par imagerie non invasive. La cellule peut, par exemple, être une cellule souche ou une cellule immunitaire. Des animaux transgéniques non humains dont le génome comprend un acide nucléique codant une séquence d'acide aminé GPCR recombinante tronquée sont également décrits. Des procédés de production de cellules souches qui expriment une GPCR recombinante tronquée, dont l'obtention d'un animal transgénique selon la présente tension et l'isolation de cellules souches à partir de l'animal transgénique, sont également décrits.
PCT/US2007/079092 2006-09-20 2007-09-20 Composés et procédés impliquant des récepteurs de recombinaison tronqués couplés à la protéine g(7) Ceased WO2008085564A2 (fr)

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EP2460542A4 (fr) * 2009-07-31 2015-02-18 Riken Marqueur des cellules endocrines du pancréas et son utilisation
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WO2017214939A1 (fr) * 2016-06-16 2017-12-21 毛侃琅 Vecteur d'expression lentiviral pour améliorer le taux d'expression du gène ccr7, et ses applications
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EP3321356A4 (fr) * 2015-07-22 2019-01-02 China Medical University Cellules souches mésenchymateuses, procédé d'amplification clonogénique associé, procédé de séparation associé et utilisations associées
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US10493162B2 (en) 2010-09-08 2019-12-03 Board Of Regents, The University Of Texas System Somatostatin receptor-based cancer therapy
EP2613791A4 (fr) * 2010-09-08 2015-05-13 Univ Texas Thérapie anticancéreuse sur la base des récepteurs de la somatostatine
US11541119B2 (en) 2010-09-08 2023-01-03 Vikas Kundra Tumor treatment methods using cells that localize to the tumor
US9731023B2 (en) 2010-09-08 2017-08-15 Board Of Regents, The University Of Texas System Somatostatin receptor-based cancer therapy
WO2012033901A2 (fr) 2010-09-08 2012-03-15 The Board Of Regents Of The University Of Texas System Thérapie anticancéreuse sur la base des récepteurs de la somatostatine
US10660918B2 (en) 2015-07-22 2020-05-26 China Medical University Method for enhancing insulin like growth factor 1 receptor expression of mesenchymal stem cell, method for isolating mesenchymal stem cell, and use of mesenchymal stem cell
US11110129B2 (en) 2015-07-22 2021-09-07 China Medical University Method for treating multiple sclerosis
US11529373B2 (en) 2015-07-22 2022-12-20 China Medical University Method for enhancing expression of insulin like growth factor 1 receptor in mesenchymal stem cell, method for obtaining mesenchymal stem cell, and method for treating brain tissue damage
EP3321356A4 (fr) * 2015-07-22 2019-01-02 China Medical University Cellules souches mésenchymateuses, procédé d'amplification clonogénique associé, procédé de séparation associé et utilisations associées
WO2017214939A1 (fr) * 2016-06-16 2017-12-21 毛侃琅 Vecteur d'expression lentiviral pour améliorer le taux d'expression du gène ccr7, et ses applications
CN106596911A (zh) * 2016-12-16 2017-04-26 石河子大学 一种证明缝隙连接在高血压炎症反应中的作用的试验方法
US12227567B2 (en) 2017-07-25 2025-02-18 Truebinding, Inc. Treating cancer by blocking the interaction of TIM-3 and its ligand
CN107604004A (zh) * 2017-09-22 2018-01-19 西安医学院 用于痘苗病毒天坛株tk基因的示踪打靶质粒及其制备方法
CN109287566A (zh) * 2018-09-20 2019-02-01 扬州大学 一种肉鹅活体估测胸肌产肉量的方法
US12497458B2 (en) 2019-01-30 2025-12-16 Truebinding, Inc. Anti-GAL3 antibodies and uses thereof
US12281166B2 (en) 2020-05-26 2025-04-22 Truebinding, Inc. Methods of treating inflammatory diseases by blocking Galectin-3
CN114703186B (zh) * 2022-03-31 2023-08-29 四川大学 肿瘤特异性启动子及其应用
CN114703186A (zh) * 2022-03-31 2022-07-05 四川大学 肿瘤特异性启动子及其应用
CN114751990B (zh) * 2022-04-01 2023-10-20 中国科学技术大学 酰胺高丝氨酸内酯受体-增强绿色荧光融合蛋白及其制备方法与应用
CN114751990A (zh) * 2022-04-01 2022-07-15 中国科学技术大学 酰胺高丝氨酸内酯受体-增强绿色荧光融合蛋白及其制备方法与应用
WO2026021448A1 (fr) * 2024-07-22 2026-01-29 健达九州(北京)生物科技有限公司 Variant de m4di et son utilisation dans le traitement de l'épilepsie

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