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WO2006023888A2 - Imagerie d'acides nucleiques cellulaires - Google Patents

Imagerie d'acides nucleiques cellulaires Download PDF

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Publication number
WO2006023888A2
WO2006023888A2 PCT/US2005/029875 US2005029875W WO2006023888A2 WO 2006023888 A2 WO2006023888 A2 WO 2006023888A2 US 2005029875 W US2005029875 W US 2005029875W WO 2006023888 A2 WO2006023888 A2 WO 2006023888A2
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WIPO (PCT)
Prior art keywords
nucleic acid
reporter
tissue
conjugate
mion
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PCT/US2005/029875
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English (en)
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WO2006023888A3 (fr
Inventor
Philip Kuocherng Liu
Young Ro Kim
Bruce R. Rosen
Christina Huang Liu
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The General Hospital Corporation
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Priority to US11/574,160 priority Critical patent/US20080118440A1/en
Publication of WO2006023888A2 publication Critical patent/WO2006023888A2/fr
Publication of WO2006023888A3 publication Critical patent/WO2006023888A3/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/06—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations
    • A61K49/18—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes
    • A61K49/1818—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes particles, e.g. uncoated or non-functionalised microparticles or nanoparticles
    • A61K49/1821—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes particles, e.g. uncoated or non-functionalised microparticles or nanoparticles coated or functionalised microparticles or nanoparticles
    • A61K49/1824—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes particles, e.g. uncoated or non-functionalised microparticles or nanoparticles coated or functionalised microparticles or nanoparticles coated or functionalised nanoparticles
    • A61K49/1827—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes particles, e.g. uncoated or non-functionalised microparticles or nanoparticles coated or functionalised microparticles or nanoparticles coated or functionalised nanoparticles having a (super)(para)magnetic core, being a solid MRI-active material, e.g. magnetite, or composed of a plurality of MRI-active, organic agents, e.g. Gd-chelates, or nuclei, e.g. Eu3+, encapsulated or entrapped in the core of the coated or functionalised nanoparticle
    • A61K49/1851—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes particles, e.g. uncoated or non-functionalised microparticles or nanoparticles coated or functionalised microparticles or nanoparticles coated or functionalised nanoparticles having a (super)(para)magnetic core, being a solid MRI-active material, e.g. magnetite, or composed of a plurality of MRI-active, organic agents, e.g. Gd-chelates, or nuclei, e.g. Eu3+, encapsulated or entrapped in the core of the coated or functionalised nanoparticle having a (super)(para)magnetic core coated or functionalised with an organic macromolecular compound, i.e. oligomeric, polymeric, dendrimeric organic molecule
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/06—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations
    • A61K49/08—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by the carrier
    • A61K49/10—Organic compounds
    • A61K49/12—Macromolecular compounds
    • A61K49/126—Linear polymers, e.g. dextran, inulin, PEG
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/06—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations
    • A61K49/18—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes
    • A61K49/1818—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes particles, e.g. uncoated or non-functionalised microparticles or nanoparticles
    • A61K49/1821—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes particles, e.g. uncoated or non-functionalised microparticles or nanoparticles coated or functionalised microparticles or nanoparticles
    • A61K49/1824—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes particles, e.g. uncoated or non-functionalised microparticles or nanoparticles coated or functionalised microparticles or nanoparticles coated or functionalised nanoparticles
    • A61K49/1827—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes particles, e.g. uncoated or non-functionalised microparticles or nanoparticles coated or functionalised microparticles or nanoparticles coated or functionalised nanoparticles having a (super)(para)magnetic core, being a solid MRI-active material, e.g. magnetite, or composed of a plurality of MRI-active, organic agents, e.g. Gd-chelates, or nuclei, e.g. Eu3+, encapsulated or entrapped in the core of the coated or functionalised nanoparticle
    • A61K49/1851—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes particles, e.g. uncoated or non-functionalised microparticles or nanoparticles coated or functionalised microparticles or nanoparticles coated or functionalised nanoparticles having a (super)(para)magnetic core, being a solid MRI-active material, e.g. magnetite, or composed of a plurality of MRI-active, organic agents, e.g. Gd-chelates, or nuclei, e.g. Eu3+, encapsulated or entrapped in the core of the coated or functionalised nanoparticle having a (super)(para)magnetic core coated or functionalised with an organic macromolecular compound, i.e. oligomeric, polymeric, dendrimeric organic molecule
    • A61K49/1863—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes particles, e.g. uncoated or non-functionalised microparticles or nanoparticles coated or functionalised microparticles or nanoparticles coated or functionalised nanoparticles having a (super)(para)magnetic core, being a solid MRI-active material, e.g. magnetite, or composed of a plurality of MRI-active, organic agents, e.g. Gd-chelates, or nuclei, e.g. Eu3+, encapsulated or entrapped in the core of the coated or functionalised nanoparticle having a (super)(para)magnetic core coated or functionalised with an organic macromolecular compound, i.e. oligomeric, polymeric, dendrimeric organic molecule the organic macromolecular compound being a polysaccharide or derivative thereof, e.g. chitosan, chitin, cellulose, pectin, starch
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/06—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations
    • A61K49/18—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes
    • A61K49/1818—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes particles, e.g. uncoated or non-functionalised microparticles or nanoparticles
    • A61K49/1821—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes particles, e.g. uncoated or non-functionalised microparticles or nanoparticles coated or functionalised microparticles or nanoparticles
    • A61K49/1824—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes particles, e.g. uncoated or non-functionalised microparticles or nanoparticles coated or functionalised microparticles or nanoparticles coated or functionalised nanoparticles
    • A61K49/1827—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes particles, e.g. uncoated or non-functionalised microparticles or nanoparticles coated or functionalised microparticles or nanoparticles coated or functionalised nanoparticles having a (super)(para)magnetic core, being a solid MRI-active material, e.g. magnetite, or composed of a plurality of MRI-active, organic agents, e.g. Gd-chelates, or nuclei, e.g. Eu3+, encapsulated or entrapped in the core of the coated or functionalised nanoparticle
    • A61K49/1866—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes particles, e.g. uncoated or non-functionalised microparticles or nanoparticles coated or functionalised microparticles or nanoparticles coated or functionalised nanoparticles having a (super)(para)magnetic core, being a solid MRI-active material, e.g. magnetite, or composed of a plurality of MRI-active, organic agents, e.g. Gd-chelates, or nuclei, e.g. Eu3+, encapsulated or entrapped in the core of the coated or functionalised nanoparticle the nanoparticle having a (super)(para)magnetic core coated or functionalised with a peptide, e.g. protein, polyamino acid
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery

Definitions

  • This invention relates to imaging cellular nucleic acids, such as imaging the delivery, uptake, and/or expression of a nucleic acid within cells in various tissues using, e.g., magnetic resonance imaging (MRI), and more particularly to MR imaging of gene expression in the brain.
  • MRI magnetic resonance imaging
  • MR imaging offers much improved spatial resolution with anatomical precision compared to other modalities such as optical imaging, computer tomography (CT), and positron emission tomography (PET).
  • CT computer tomography
  • PET positron emission tomography
  • the common goal is to deliver a suitable contrast agent or label to the relevant tissue, and more specifically into the cells.
  • a suitable contrast agent or label In the brain, for example, one must typically find a way to overcome the blood-brain-barrier.
  • most of the known contrast agents for example, for MRI, have limited permeability to cells when administered to live subjects, and as a result the limited permeability provides only a short and often unstable window for MR imaging.
  • the invention is based, in part, on the discovery that short nucleic acid sequences, e.g., phosphorothioated nucleic acid sequences, can be linked to one or more reporter groups to form reporter conjugates, which transport the reporter groups into cells, to cell membranes, or into the vicinity of cells in which a specific cellular nucleic acid, e.g., an RNA, DNA, gene, or chromosome, without the need for translocation sequences or the like.
  • Liposomes may aid the uptake of the reporter conjugates into cells.
  • the new reporter conjugates can be used to image expression of target cellular nucleic acids non-invasively in a variety of tissues, such as tissues of the brain, liver, pancreas, heart, lung, spinal cord, prostate, breast, gastrointestinal tract, ovary, and kidney.
  • the reporter group can be an MRI contrast agent, such as a paramagnetic label, such as a superparamagnetic iron oxide particle whose maximum diameter is between 1 nm and 2000 nm, e.g., between 2 nm and 1000 nm. In some embodiments, the maximum particle diameter is between 10 nm and 100 nm.
  • the particle can be attached to the targeting nucleic acid through entrapment in a cross-linked dextran.
  • the paramagnetic label is a chelated metal such as Gd 3+ or Dy 3+ .
  • the reporter group can also be a fluorescent label, e.g., FITCs, Texas Red, or Rhodamine.
  • the invention features reporter conjugates for imaging cellular nucleic acids that, include a single targeting nucleic acid linked to one or more reporter groups. These targeting nucleic acids and reporter groups are described in detail herein. In-another-aspect ⁇ hejnvention-feamres ⁇ methods-ofimagmg-axellularjiucleic- acid in a tissue in vivo.
  • the methods include obtaining a reporter conjugate including a targeting nucleic acid linked to a reporter group, wherein the targeting nucleic acid hybridizes to a target nucleic acid molecule corresponding to the cellular nucleic acid to be imaged; administering the reporter conjugate to the tissue in an amount sufficient to provide a detectable image; allowing sufficient time to pass to allow any unbound reporter conjugate to leave the tissue; and imaging the tissue, wherein a detectable image of the reporter group in the tissue indicates the presence of the cellular nucleic acid.
  • the target nucleic acid molecule can include a messenger RNA transcribed from a target gene, and the targeting nucleic acid can include an anti-sense strand that hybridizes to a portion of the messenger RNA, wherein the presence of the cellular nucleic acid indicates expression of the target gene.
  • the target gene can be a therapeutic gene previously delivered to the tissue.
  • the tissue can be, e.g., brain, heart, lung, liver, pancreas, spinal cord, prostate, breast, gastrointestinal system, ovary, or kidney tissue.
  • the tissue can be in a patient, e.g., a human patient.
  • the reporter group can be a superparamagnetic iron oxide particle whose maximum diameter is between 1 nm and 2000 nm.
  • the reporter conjugate can be administered by, e.g., intravenous injection or intra-cerebro ventricular infusion.
  • the invention features reporter conjugates for imaging cellular nucleic acids that include a single targeting nucleic acid linked to one or more superparamagnetic iron oxide particles whose maximum diameter is between 1 nm and 1000 nm (e.g., between 10 and 100 nm).
  • the particles are a monocrystalline iron oxide nanoparticle (MION), ultra small superparamagnetic iron oxide particle (USPIO), or cross-linked iron oxide (CLIO) particle.
  • MION monocrystalline iron oxide nanoparticle
  • USPIO ultra small superparamagnetic iron oxide particle
  • CLIO cross-linked iron oxide
  • the invention features methods of imaging target cells (e.g., cornu ammonis neurons) that are undergoing or have undergone programmed cell death in a tissue.
  • the methods include obtaining a reporter conjugate including a targeting nucleic acid linked to a reporter group, wherein the targeting nucleic acid hybridizes to a target nucleic acid molecule corresponding to the target cells; administering the reporter conjugate to the tissue in an amount sufficient to provide a detectable image; allowing sufficient time to pass to allow any unbound reporter conjugate to leave the tissue; and imaging the tissue, wherein a presence of a detectable image of the reporter -group-in-the tissue -indieates-that-the-eells-in-the-tissue-have-not-undergone- programmed- cell death, and an absence of a detectable image of the reporter group indicates that the cells are undergoing or have undergone programmed cell death.
  • the invention features methods of treating a disorder, e.g., a cancer, in a patient.
  • the methods include obtaining a conjugate including a targeting nucleic acid linked to a therapeutic agent and a reporter group, wherein the targeting nucleic acid hybridizes to a target nucleic acid molecule corresponding to a target organ or tissue; and administering the conjugate to a patient in an amount sufficient to treat the disorder.
  • the targeting nucleic acid preferentially binds to an oncogene or a mutant mRNA transcribed by an oncogene.
  • the invention features the use of a reporter conjugate including a targeting nucleic acid linked to a reporter group, wherein the targeting nucleic acid hybridizes to a target nucleic acid molecule corresponding a cellular nucleic acid, in the preparation of a pharmaceutical composition for imaging a cellular nucleic acid in a tissue in vivo
  • the invention features methods of imaging expression of a target gene in a tissue in vivo, by obtaining a reporter conjugate including a targeting nucleic acid linked to a reporter group, wherein the targeting nucleic acid hybridizes to a target nucleic acid molecule corresponding to the target gene whose expression is to be imaged; administering the reporter conjugate to the tissue in an amount sufficient to provide a detectable image; allowing sufficient time to pass to allow any unbound reporter conjugate to leave the tissue; and imaging the tissue, wherein a detectable image of the reporter group in the tissue indicates that the target gene has been expressed.
  • the invention features methods of imaging a cellular nucleic acid in a tissue by obtaining a reporter conjugate including a targeting nucleic acid linked to a reporter group, wherein the targeting nucleic acid hybridizes to a target nucleic acid molecule corresponding to the cellular nucleic acid to be imaged; administering the reporter conjugate to the tissue in an amount sufficient to provide a detectable image; allowing sufficient time to pass to allow any unbound reporter conjugate to leave the tissue; and imaging the tissue, wherein a detectable image of the reporter group in the tissue indicates the presence of the cellular nucleic acid.
  • the invention also includes methods of treating a cancer cell in a patient by obtaining a conjugate -including a targeting-nucleic acid-linked-to-an -anti-cancer agent— wherein the targeting nucleic acid hybridizes to a target nucleic acid molecule corresponding to the cancer cell; and administering the conjugate to the patient in an amount sufficient to inhibit growth of the cancer cell.
  • the conjugate can further include a reporter group.
  • the invention also includes methods of treating a disorder in a patient by obtaining a conjugate including a targeting nucleic acid linked to a therapeutic agent, e.g., a dextran-coated therapeutic agent, wherein the targeting nucleic acid hybridizes to a target nucleic acid molecule corresponding to a desired target organ or tissue, and administering the conjugate to the patient in an amount sufficient to treat the disorder.
  • a conjugate including a targeting nucleic acid linked to a therapeutic agent, e.g., a dextran-coated therapeutic agent, wherein the targeting nucleic acid hybridizes to a target nucleic acid molecule corresponding to a desired target organ or tissue, and administering the conjugate to the patient in an amount sufficient to treat the disorder.
  • the conjugate can further include a reporter group.
  • the invention includes methods of decreasing expression of a gene in a cell and, optionally, imaging a cellular nucleic acid by obtaining a reporter conjugate including a nucleic acid, e.g., a phosphorothioated nucleic acid (e.g., a phosphorothioated RNA), that decreases (e.g., is designed to decrease) expression of a target gene, and administering the conjugate to a cell in an amount sufficient to decrease expression of the target gene, and, optionally, allowing sufficient time to pass to allow any unbound reporter conjugate to leave the tissue; and imaging the tissue.
  • the nucleic acid can be, e.g., an antisense nucleic acid, a short inhibitory RNA (siRNA), a micro RNA (miRNA), or a double stranded RNA (dsRNA).
  • the invention also includes methods of imaging (e.g., visualizing or locating) a cell type that expresses a gene in a subject.
  • the methods include obtaining a conjugate including a targeting nucleic acid linked to a reporter group, wherein the targeting nucleic acid hybridizes to a target nucleic acid molecule that is expressed by the cell type to be imaged, administering the conjugate to a subject in an amount sufficient to produce a detectable image, and imaging the tissue, wherein the presence of the conjugate is indicative of the cell type.
  • the cell type to be imaged can be, e.g., a cancer cell, a transgenic cell, or a stem cell (e.g., an embryonic stem cell).
  • the invention includes use of a reporter conjugate including a targeting nucleic acid linked to a reporter group, wherein the targeting nucleic acid hybridizes to a target nucleic acid molecule corresponding to a cellular nucleic acid, in the preparation of a pharmaceutical composition for imaging a cellular nucleic acid in a tissue in vivo.
  • the reporter conjugate can further include a therapeutic -agent—
  • a nucleic acid that hybridizes or binds "specifically" to a target nucleic acid hybridizes or binds preferentially to the target, and does not substantially bind to other molecules or compounds in a biological sample.
  • magnet means having positive magnetic susceptibility and lacking magnetic hysteresis (ferromagnetism).
  • an "oncogene” is an allele of a gene that is associated with increased risk of cancer, e.g., a mutant form of a proto-oncogene or tumor suppressor gene, or a viral oncogene. Numerous examples of oncogenes are known in the art (see, e.g., Vogelstein and Rinzler, Nat. Med., 10:789-99 (2004)) The new conjugates and methods allow real time imaging, such as MRI, and avoid the need for biopsies. The imaging is safe and can be performed as often as is needed for several days.
  • FIGs. lAto IH are a series of schematic representations of reporter conjugates, showing certain possible attachments of reporter groups, such as contrast agents or labels, that can be linked, e.g., via covalent bonds, directly or indirectly to the 5' (FIGs. lA and 1C) or 3' terminus (FIGs. IB and ID), individually (FIGs.
  • reporter groups such as contrast agents or labels
  • reporter groups e.g., contrast agents and labels that can be used include, but are not limited to, paramagnetic agents, fluorescent labels (FITC, rhodamine, Texas Red), radioactive isotopes, individually or combinations. More than 50 unique reporter groups can be made in an average length (2 kilobases) of a gene transcript (mRNA). For example, 50 different reporter conjugates can be made that specifically bind to a specific target nucleic acid, e.g., to different portions of the same target.
  • FITC fluorescent labels
  • rhodamine Texas Red
  • radioactive isotopes individually or combinations.
  • More than 50 unique reporter groups can be made in an average length (2 kilobases) of a gene transcript (mRNA). For example, 50 different reporter conjugates can be made that specifically bind to a specific target nucleic acid, e.g., to different portions of the same target.
  • AU 50 conjugates can have the same or different reporter groups, and could have different (e.g., up to 50 different) reporter groups on the 50 different conjugates. This can be used to provide signal amplification. In addition, similar numbers of reporter contrast agents can be made to the exons of a given gene.
  • FIG. II is a legend depicting the symbols used in FIGs. IA to IH.
  • FIGs. 3 A to 3C are micrographs that show the presence of iron oxide (MION stained with Prussian blue and tissue was counterstained for nuclei using Nuclear Fast Red) in cells at the same regions of different animals shown in the MRI (FIG. 2) 11 days after infusion of the reporter MION-A26 (SEQ ID NO:1).
  • FIGs. 1 iron oxide
  • FIG. 3B and 3C are inserts from FIG. 3 A that show that iron oxide was present around and within the nuclei in the cortex (3B) and CA neurons (3C).
  • FIG. 3A shows the corpus callosum. Arrows show the presence of iron oxide in perivascular spaces that connect the cerebral ventricular space to brain cells. The bar in each micrograph indicates 20 micrometer ( ⁇ m).
  • FIG. 4 is a fluorescence micrograph that shows the distribution of fiuorescent- ODN in the brain (as condensed bright dots) after infusion of the reporter conjugate MION- A26 (SEQ ID NO:l)-digoxigenin (detected using fluorescein isothiocyanate [FITC] labeled IgG against digoxigenin).
  • the distribution of A26 (SEQ ID NO:1) is present throughout the brain and tissue sample from -1.4 mm to the Bregma.
  • the A26 used in this study contains a sequence that is complementary to c-fos mRNA and has been able to detect c-fos mRNA expression in the brain using in situ hybridization (see below in FIG.
  • FIG. 5 is an autoradiograph that shows three consecutive samples of c-fos mRNA detected using conventional assay in situ hybridization (radioactive antisense A26- 35 S) in a postmortem mouse brain.
  • FIG. 6 is a micrograph of c-fos mRNA in mouse hippocampus using molecular biological assay in another postmortem mouse brain by using the techniques of in situ reverse transcription PCR with and A26 labeled with digoxigenin, followed by detection using alkaline phosphatase-labeled antibody against digoxigenin.
  • FIG. 7 shows a serial T2* map of mouse brain that received a MION-s-ODN reporter conjugate immediately ( ⁇ 30 minutes; top row) and one day (bottom row) post 5 infusion from the same subject.
  • T2* maps of contiguous 0.5 mm MR slices from selected posterior to anterior portion of the brain (five contiguous slices, -1.5 to —3.5 mm to the bregma) are shown.
  • the presence of MION results in regions of signal reduction (therefore, decreased T2*, arrows) that are apparent one day after infusion.
  • the persistent signal reduction within the ventricles, indicative of MION presence, can still be observed (FIG. 8A) at day 1 after infusion of MION-A26 (SEQ ID NO:1).
  • FIG. 8B shows no retention of MION in live animals that received a mixture of unconjugated 5 MION and s-ODN. The MION signal dissipated within three hours.
  • FIGs. 9 A to 9B shows MR images of animals, 30 minutes and 3 hours after infusion of MION-dextran (9A) (a control that shows no retention) or MION-dUTP (9B) (another control that also shows no retention).
  • FIGs. 1OA to 1OC are bar graphs (1OA and 10B) and MR images of mouse brain 0 (10C).
  • FIGs. 1OA and 1OB compare R2* (1/T2*) values in contralateral cortical regions (boxes in FIG. 10C) from selected brain slices of mice injected with MION- A26 (SEQ ID NO:1) and MION immediately ( ⁇ 30 minutes, 10A) or 1 day after infusion (10B).
  • the bar graph shows that the brain cells retained significant amounts of the MION reporter conjugate, and that it was distributed from the left ventricle 5 (infusion site) to the right ventricle and the cortex.
  • FIGs. 1 IA and 1 IB are MR images of an animal brain that received MION- -A26-(SEQ-ID N ⁇ :-l-) ⁇ -The-images-were-aequired at -three-days-after -the-infusion.-The- distribution of MION can be seen in the cerebellum (FIG. HA; in vivo MRI, sagittal view) and in the white matter of the spinal cord (FIG. HB, ex vivo MRI). 0 FIG.
  • the image shows a homogenous retention of reporter MION- A26 (SEQ ID NO: 1).
  • Signal reduction can be observed in areas such as the cortex, white matter tracks, hippocampus and the striatum.
  • FIGs. 13Ato 13D are a series of fluorescence micrographs of a rat brain that show the presence of s-ODN (from an infusion of the reporter conjugate MION- A26 (SEQ ID NO:l)-digoxigenin) as clustered bright dots in the olfactory lobe (13A), the cerebellum (13B) and the cortex (13C).
  • FIG. 13D is a control that shows background fluorescence in a sample without a fluorescent antibody label.
  • the arrows in FIG. 13B denote Purkinje cells.
  • FIGs. 14A and 14B are two schematic representations of reporter conjugates that can be used for therapeutic purposes, e.g., to treat cancer, when a target gene contains a known mutation.
  • FIG. 14C is a legend depicting the symbols used in FIGs. 14A and 14B.
  • FIGs. 15A and 15B are micrographs that show a preferential distribution of digoxigenin-labeled A26 (anti-sense to c-fos mRNA) in glioblastoma in rats.
  • Cells of the glioblastoma cell line D74 (ENU-induced, 10 5 each rat) were implanted to the cortex of a Fisher 344 rat.
  • digoxigenin labeled s-ODN (1 nmol) was infused via ICV route on the contralateral hemisphere.
  • the reporter conjugate remains in the glioblastoma cells as indicated by HE staining (15A) and by fluorescence of FITC-labeled antibodies to digoxigenin (15B).
  • FIG. 16 is a micrograph of animal brain showing early programmed cell death
  • FIGs 17A to 17D are fluorescent micrographs of in situ RT-PCT on postmortem mouse brain samples.
  • MION-A26 SEQ ID NO:1 was used for reverse transcription.
  • MION-Ran SEQ ID NO:3 was used for reverse transcription.
  • FIG 17E depicts the negative control without PCR primers-
  • FIGs. 18A and 18B are bar graphs depicting R2* values of brain regions in vivo.
  • FIG. 18A depicts MION signal in (as elevated R2*) the contralateral cortices on days 1 and 2 following infusion with MION-A26 or MION-Ran.
  • FIG. 18B depicts MION signal in the contralateral hemisphere two days after global cerebral ischemia induced using bilateral common carotid arteries occlusion (BCCAO) and infusion with MION- A26 or MION-Ran.
  • FIGs. 19A and 19B are c-fos mRNA expression maps of mouse brains produced by R2* map computed from ex vivo MR microscopy. Brightness indicates increased signal intensity.
  • FIGs. 19C to 19G are c-fos mRNA expression maps of mouse brains produced by in situ hybridization. In situ hybridization was performed on mice without BCCAO (19C) and 30, 60, 120, and 240 minutes following a transient 30-minute BCCAO and reperfusion (19D-19G).
  • FIG. 19H is a schematic map of C57bl6 mouse showing brain regions where elevated c-fos mRNA is expressed (Paxinos and Franklin, The Mouse Brain in Stereotaxic Coordinates, Academic Press Limited, London, 2001).
  • Ag amygdala
  • TH thalamus
  • HT hypothalamus
  • Hip hippocampus
  • Pic piriform cortex.
  • FIG. 2OA is a beta-actin mRNA map of mouse brains produced by R2* map (acquired similar to FIGs. 19A and 19B). Brightness indicates increased signal intensity.
  • TH thalamus
  • HT hypothalamus
  • Hip hippocampus.
  • FIG. 2OB is a c-fos mRNA expression map of mouse brain produced by in situ hybridization. In situ hybridization was performed on mice without BCCAO.
  • FIG. 2OC is a bar graph depicting R2* values of brain regions of normal animals in vivo one day following infusion with MION-A26 (c-fos) or MION-BA25A1 (ACGCAGCTCAGTAACAGTCCGCCTA O(SEQ ID NO:6); Alonso et al., J. MoI. Evol., 23:11-12, 1986).
  • FIG. 21 A is a bar graph depicting R2* from hippocampus in live animals with or without BCCAO and infused with MION-ODN.
  • FIG. 2 IB and 21C are MR images of MION-ODN retention in the hippocampus of normal (21B) or stroke model (21C) mice.
  • JlheinventioriJcelatesioj ⁇ ew-methods.andx.omp ⁇ sitionsJbrLimagingJhe. uptake/distribution and/or expression of specific target genes, such as therapeutic genes, in various cells and tissues, such as in the brain, non-invasively using various imaging modalities, such as MRI.
  • reporter conjugates after delivery to live subjects, can be internalized by brain cells (FIGs 2-3); the cells that have retained the MION and ODN (FIG 4) express c-fos messenger RNA, and the labeled antisense ODN can detect its expression (FIGs. 5-6). Then the examples will show that the reporter conjugate MION-s-ODN can be used to produce MR images of the MION and ODN in the brain in live subjects (FIGs. 7-12).
  • the new imaging methods use novel reporter conjugates to image the uptake and distribution of targeting nucleic acids, e.g., oligodeoxyribonucleotides (ODN), delivered to the brain or other tissues in live animals and humans.
  • the conjugates include a reporter group, such as a contrast agent or a label, e.g., an MRI contrast agent, e.g., iron oxide nanoparticles (e.g., MION-dextran) linked to a targeting nucleic acid (such as a single-stranded ODN) that hybridizes to a portion of a particular target nucleic acid molecule.
  • the conjugate is delivered to the tissue containing, or thought to contain, a target gene, whose uptake, distribution, or expression is to be imaged.
  • a target gene whose uptake, distribution, or expression is to be imaged.
  • the reporter conjugate is to be delivered to the brain
  • convection-enhanced delivery to the cerebral ventricles such as to the lateral ventricle (Liu et al., 1994 and Cui et al., 1999) or the 4 th ventricles (Sandberg et al., J. Neuro- Oncology, 58:187-192, 2002). Delivery can also be intrathecal (Liu et al. (2004) Magn. Reson. Med. 51 : 978-87) or by any additional routes that lead directly or indirectly to brain cells.
  • the tissue is imaged.
  • the tissue can be imaged with a series of high-resolution T2*-weighted MR images, e.g., taken 1, 2, or 3 days after infusion of the reporter conjugate.
  • To-use-the-new-Gonjugates-and-methods-to-image-gene-expressionj-the-targeting- nucleic acid can be prepared as an anti-sense strand that is designed to hybridize to a portion of a target messenger RNA transcribed from the target gene.
  • a reporter conjugate including this anti-sense strand is detected in cells in a tissue, it provides a clear indication that that target mRNA is present in the cell, and thus that the target gene is being expressed.
  • Guidance on designing nucleic acids that hybridize to a target under specific conditions e.g., intracellular conditions
  • the new reporter conjugates and imaging methods open a new venue to detect and track the delivery and uptake of nucleic acid molecules in live animals for neuroscience research and various clinical applications.
  • the reporter conjugates are prepared by conjugating or linking one or more targeting nucleic acids to one or more reporter groups, such as magnetic particles that change the relaxivity of the cells once internalized so that they can be imaged using MRI.
  • One targeting nucleic acid can have multiple (e.g., 2, 3, or more) reporter groups attached (all or some the same or different), or a set of numerous reporter conjugates can be created in which they all have the same targeting nucleic acid and 2 or more different reporter groups within the set.
  • a set of reporter conjugates can be made that have different targeting nucleic acids that all target different portions of the same target gene (or that target different target genes), and each have the same or different reporter groups.
  • nucleotides also referred to herein as an oligonucleotide or ODN
  • reporter-groupSj- such-as a-contrast agentj-linked-to-either-the-S— or-3— ends of-the-QDN,- either directly, e.g., by a covalent bond or via an optional linker group or "bridge” (e.g., a linkage of a desired length) between the ODN and the reporter group(s).
  • the targeting nucleic acid must have at least 80% sequence homology (identity) with a sequence that is complementary to a portion of the target nucleic acid molecule.
  • the targeting nucleic acid can be either single-stranded DNA or RNA, and is typically an anti-sense strand, and thus complementary, to a portion of the target nucleic acid.
  • the ODN may include one or multiple internal sites that can be attached to a reporter group, e.g., labeled, for example, with a radioactive or fluorescent label.
  • FIGs. IE to IH show reporter conjugates that include two or more reporter groups, as well as an optional antibody that can be attached at either end of the molecule (FIGs. IG and IH).
  • These antibodies are typically ones that bind specifically to cell-surface antigens of particular cells or cell types to direct the reporter conjugate to the appropriate cells.
  • the reporter conjugates Once on the surface of the cell, the reporter conjugates pass through the cell membrane and into the cells, thereby delivering the reporter group into the cell.
  • the targeting nucleic acids hybridize preferentially to their specific target nucleic acid, such as an mRNA, and remain bound within the cell. Absent the targeting nucleic acid, the reporter groups are not retained within the cells.
  • the targeting nucleic acid can be linked to the reporter group or groups by a variety of methods, including, e.g., covalent bonds, bifunctional spacers ("bridge") such as, avidin-biotin coupling, Gd-DOP A-dextran coupling, charge coupling, or other linkers.
  • bridge bifunctional spacers
  • the reporter groups can be contrast agents such as magnetic particles, such as superparamagnetic, ferromagnetic, or paramagnetic particles.
  • Paramagnetic metals e.g., transition metals such as manganese, iron, chromium, and metals of the lanthanide group such as gadolinium
  • the particle size can be between 1 nm and 2000 nm, e.g., between 2 nm and 1000 ran (e.g., 200 or 300 nm), or between 10 nm and 100 nm, as long as they can still be internalized by the cells.
  • the magnetic particles are typically nanoparticles.
  • particle size is controlled, with variation in particle size being limited, e.g., substantially all of the particles having a diameter in the range of about 30 nm to about-50 nm.
  • An individual particle can consist of a single metal oxide crystal or a multiplicity of crystals.
  • contrast agents useful for MR imaging Tl and T2 agents.
  • Tl agent such as manganese and gadolinium
  • T2 agent such as manganese and gadolinium
  • T2 agent such as iron
  • T2 spin-spin transverse relaxation time
  • Optimal MRI contrast can be achieved via proper administration of contrast agent dosage, designation of acquisition parameters such as repetition time (TR), echo spacing (TE) and RF pulse flip angles.
  • acquisition parameters such as repetition time (TR), echo spacing (TE) and RF pulse flip angles.
  • MIONs monocrystalline iron oxide nanoparticles as described, e.g., in U.S. Patent No. 5,492,814; Whitehead, U.S. Patent No.
  • These particles can also be superparamagnetic iron oxide particles (SPIOs), ultra small superparamagnetic iron oxide particles (USPIOs), and cross-linked iron oxide (CLIO) particles (see, e.g., U.S. Patent No. 5,262,176).
  • SPIOs superparamagnetic iron oxide particles
  • USPIOs ultra small superparamagnetic iron oxide particles
  • CLIO cross-linked iron oxide
  • MIONs can consist of a central 3 nm monocrystalline magnetite-like single crystal core to which are attached an average of twelve 10 kD dextran molecules resulting in an overall size of 20 nm (e.g., as described in U.S. Patent No. 5,492,814 and in Shen et al., "Monocrystalline iron oxide nanocompounds (MION): Physicochemical Properties," Magnetic Resonance in Medicine, 29:599-604 (1993), to which nucleic acids can be conjugated for targeted delivery.
  • the dextran/Fe w/w ratio of a MION can be 1.6: 1.
  • At room temperature relaxivity in an aqueous solution at room temperature and 0.47 Tesla can be: Rl ⁇ 19/mM/sec, R2 ⁇ 41/mM/sec.
  • MIONs elute as a single narrow peak by high performance liquid chromatography with a dispersion index of 1.034; the median MION particle diameter (of about 21 nm as measured by laser light scattering) corresponds in size to a protein with a mass of 775 kD and contains an average of 2064 iron molecules.
  • the physicochemical and biological properties of the magnetic particles can be improved by crosslinking the dextran coating of magnetic nanoparticles to form CLIOs to inerease blood-half-lifeand-stabilily-of-the-reporter-Gomplex— -tte-Gross-lmked-dextran- coating cages the iron oxide crystal, minimizing opsonization. Furthermore, this technology allows for slightly larger iron cores during initial synthesis, which improves the R2 relaxivity.
  • CLIOs can be synthesized by crosslinking the dextran coating of generic iron oxide particles (e.g., as described in U.S. Patent No. 4,492,814) with epibromohydrin to yield CLIOs as described an U.S. Patent No. 5,262,176.
  • the magnetic particles can have a relaxivity on the order of 35 to 40 mM/sec, but this characteristic depends upon the sensitivity and the field strength of the MR imaging device.
  • the relaxivities of the different reporter conjugates can be calculated as the slopes of the curves of 1/Tl and 1/T2 vs. iron concentration; Tl and T2 relaxation times are determined under the same field strength, as the results of linear fitting of signal intensities from serial acquisition: (1) inversion-recovery MR scans of incremental inversion time for Tl and (2) SE scans of a fix TR and incremental TE. Stability of the conjugates can be tested by treating them under different storage conditions (4 0 C, 21 0 C, and 37 0 C for different periods of time) and performing HPLC analysis of aliquots as well as binding studies.
  • the paramagnetic label on the probe is a metal chelate.
  • Suitable chelating moieties include macrocyclic chelators such as 1,4,7,10- tetrazazcyclo-dodecane-N,N',N",N'"-tetraacetic acid (DOTA).
  • DOTA 1,4,7,10- tetrazazcyclo-dodecane-N,N',N",N'"-tetraacetic acid
  • Gd 3+ gadolinium
  • Dy 3+ dysprosium
  • europium are suitable.
  • Manganese can also be used for imaging tissues other than in the brain.
  • CEST Chemical Exchange Saturation Transfer
  • the CEST method uses endogenous compounds such as primary amines as reporter groups that can be linked to the ODN.
  • reporter groups are labels such as near infrared molecules, e.g., indocyanine green (ICG) and Cy5.5 and quantum dots, which can be linked to the targeting nucleic acid and used in optical imaging techniques, such as diffuse optical tomography (DOT) (see, e.g., Ntziachristos et al., Proc. Natl. Acad. Sci. USA, 97:2767- 2773, 2000).
  • Fluorescent labels such as FITCs, Texas Red, and Rhodamine can also be linked to the targeting nucleic acid.
  • Radionuclides such as 11 C, 13 N or 15 O, can be synthesized into the targeting nucleic acids to form the reporter conjugates.
  • radiopharmaceuticals such as radiolabeled tamoxifen (used, e.g., for -breast- can6er-ehemotherapy)-and-radiolabeled-antibodies-can-be-used ⁇ -Eor-example,- they can be coated with dextran for attachment to the targeting nucleic acids as described herein.
  • radio-conjugates have application in positron emission tomography (PET).
  • PET positron emission tomography
  • Radioisotopes such as 32 P, 33 P, 35 S (short half-life isotopes) (Liu et al. (1994) Ann.
  • radioactive iodine, and barium can also be integrated into or linked to the targeting nucleic acid to form conjugates that can be imaged using X-ray technology.
  • two or more reporter groups, of the same or different kinds, can be linked to a single targeting nucleic acid.
  • the targeting nucleic acids are typically single-stranded, anti-sense oligonucleotides of 12, 15, 18, 20, 23, 25, 26, and up to 30 nucleotides in length. They are designed to hybridize to the target gene (if present in sufficient numbers in a cell), or to hybridize to a messenger RNA transcribed from the gene whose expression is to be imaged. They can be protected against degradation, e.g., by using phosphorothioate, which can be included during synthesis. In addition, by keeping the length to 30 or fewer nucleotides, the non-specific nuclease/protease response that could destroy cellular mRNA and induce a cytotoxic reaction can be avoided.
  • the reporter group and the targeting nucleic acid are then linked to produce the reporter conjugate using any of several known methods.
  • the contrast agent is a MION
  • this molecule can be linked to a nucleic acid by phosphorothioating the oligonucleotide and labeling it with biotin at the 5' end.
  • the dextran coated MION can be activated and conjugated to the biotin-labeled oligonucleotide using avidin based linkers, such as NeutrAvidin ® (Pierce Chem.).
  • liposomes, lipofectin, and lipofectamine can be used to help get the entire conjugate into a cell.
  • a reporter conjugate can be diluted in a physiologically acceptable fluid such as buffered saline, dextrose or mannitol.
  • a physiologically acceptable fluid such as buffered saline, dextrose or mannitol.
  • the solution is isotonic.
  • the conjugate -ean-be-lyophilized-and-reGonstituted-before-injection-w-ith a-physiolog-ical-fluid.-Ihe conjugate can be administered parenterally, e.g., by intravenous (IV) injection, subcutaneous injection, or intra-muscular administration, depending on the tissue to be imaged.
  • IV intravenous
  • a useful route of administration is the intracerebroventricular (ICV) route.
  • the conjugate When administered intravenously, the conjugate can be administered at various rates, e.g., as rapid bolus administration or slow infusion.
  • useful dosages are between about 0.1 and 10.0 mg of iron per kg, e.g., between 0.2 and 5 mg/kg for a 1.5 Tesla medical scanner.
  • there is a field dependence component in determining the contrast dosage Doses of iron higher than 10 mg/kg should be avoided because of the inability of iron to be excreted.
  • These types of contrast agents can be used at a dosage of 0.001 to 0.1 mg/kg body weight for ICV administration in the rodents.
  • the dose When administered by IV injection and chelated gadolinium is used as the paramagnetic label, the dose will be between 10 ⁇ moles and 1000 ⁇ moles gadolinium/kg, e.g., between 50 and 500 ⁇ moles gadolinium/kg. Doses above 1000 ⁇ moles/kg produce hyperosmotic solutions for injection.
  • the new reporter conjugates will shorten the relaxation times of tissues (Tl and/or T2) and produce brightening or darkening (contrast) of MR images of cells, depending on the tissue concentration and the pulse sequence used.
  • T2 weighted pulse sequences and when iron oxides are used darkening will result.
  • Tl weighted pulse sequences and when gadolinium chelates are used brightening will result.
  • Contrast enhancement will result from the selective uptake of the conjugate in cells that contain the target gene.
  • paramagnetic metal chelate-type probes will show renal elimination with uptake by the liver and spleen, and to a less degree by other tissues.
  • Superparamagnetic iron oxide crystal-type probes are too large for elimination by glomerular filtration. Thus, most of the administered probe will be removed from the blood by the liver and spleen.
  • Superparamagnetic iron oxides are biodegradable, so the iron eventually will be incorporated into normal body iron stores.
  • Various reporter groups for medical imaging are routinely administered to patients intravenously, but can also be delivered by intra-peritoneal, intravenous, or -intra-arterial-injection-- All-of-these-methods-ean-deli-ver-the-new-reporter-conjugates- throughout the body except to the brain due to the existence of the blood brain barrier (BBB).
  • BBB blood brain barrier
  • MR imaging can be performed in live animals or humans using standard MR imaging equipment, e.g., clinical, wide bore, or research oriented small-bore MR imaging equipment, of various field strengths.
  • Imaging protocols typically consist of Ti, T 2 , and T 2 * weighted image acquisition, Tl weighted spin echo (SE 300/12), T2 weighted SE (SE 5000/variable TE) and gradient echo (GE 500/variable TE or 500/constant TE/variable flip angles) sequences of a chosen slice orientation at different time points before and after administration of the reporter conjugate.
  • biodistribution studies and nuclear imaging can be carried out using excised tumors of animals that have received a single dose of labeled reporter complex, e.g., MION-s-ODN.
  • the same assay can be used to analyze the biodistribution of other new reporter conjugates.
  • animals receive an infusion of the conjugate. After injection, differences in R2* maps (inverse of T2* maps) are determined after a pre-defined period of time. If significant, the reporter conjugate can be used in clinical imaging of that specific transgene.
  • Biodistribution studies can be used to show a higher concentration of the reporter conjugate in cells expressing the target gene compared to matched cells that do not express (or over-express) the target gene in the same animal.
  • This image evaluation technique can also applied to other imaging modalities such as PET, X-ray, and DOT, in which radionuclides, radioisotopes, and/or fluorescent probes are detected.
  • imaging modalities such as PET, X-ray, and DOT, in which radionuclides, radioisotopes, and/or fluorescent probes are detected.
  • imaging modalities and their corresponding reporter groups, are described in Min et al. (Gene Therapy, 11:115-125 (2004)).
  • the new methods and compositions have numerous practical applications.
  • the availability of reporter conjugates to image cellular nucleic acids, e.g., to image, gene expression, is important for monitoring gene therapy where exogenous genes are introduced to ameliorate a genetic defect or to add an additional gene function to cells.
  • the new methods can also used to image endogenous gene expression during development and/or pathogenesis of disease.
  • the new compositions can be used to develop an animal line that has a target gene under the control of a given promoter under study, so that promoter activity can be directly visualized.
  • the new methods can also be used for imaging gene expression in deep organs using MR imaging, and for imaging tumors that over-express certain target genes compared to normal cells.
  • the new reporter conjugates can be used for in vivo monitoring of gene expression. This will have direct applications in determining efficacy and persistence of gene therapy by non-invasive imaging and imaging gene expression over time in the same subject.
  • the new methods will also be useful in testing many of the anticipated new vectors that are currently being designed in an effort to create safer and more efficient gene delivery systems.
  • there are a number of strategies to improve viral gene delivery to brain tumors either by modifying the blood-brain-barrier (BBB) or by targeting viruses. Irrespective of the strategy, methods that can quantitate delivery and follow gene expression over time are necessary tools in the development of gene therapy.
  • BBB blood-brain-barrier
  • the new reporter conjugates and methods can also be used to image knockdown -gene-products-that- may-be-harmful- to-normal-brain-function.-
  • the mutant ODN can be synthesized to be complementary to a mutated oncogene, and can be designed to carry one or more anti-cancer agents, such as radiopharmaceuticals or radioisotopes that can inhibit or kill the cancer cell (see FIGs. 14A to 14C). Furthermore, the ability of the reporter conjugate to discriminate between the mutant copy and wild-type copy of a target mRNA transcribed from an oncogene can be used to enable the reporter conjugate to preferentially bind to the mutant mRNA, thereby inhibiting translation of the mutant mRNA into a gene product, and thus inhibit expression of the mutant oncogene.
  • anti-cancer agents such as radiopharmaceuticals or radioisotopes that can inhibit or kill the cancer cell
  • cancer cells are known to have higher abilities of endocytosis, which allows cancer cells to take up extracellular particles such the MION-s-ODN reporter conjugate.
  • the s-ODN that was used in the reporter conjugate had a higher affinity to glioblastoma cells than to normal neurons. This preferential ability to bind to the glioblastoma cells can be used to deliver cancer therapeutics via direct or indirect linkage, with or without tumor-specific antibody, on the reporter contrast agent.
  • the new reporter conjugates 14A and 14B can be used for detection, diagnosis, and therapy, e.g., for treating cancers such as brain tumors.
  • the new reporter conjugates can also be used to image apoptosis (programmed cell death, PCD) (See Example 12).
  • PCD programmed cell death
  • FIG. 16 shows that early PCD can be detected in CA neurons using TUNEL (te ⁇ ninal UTP nick end labeling) in postmortem brain tissue after global stroke in mice. PCD starts with self-destruction of nuclear DNA (DNA fragmentation is detected using the TUNEL assay), and then other components of the cell; dead neurons are removed and are generally not immediately replaced.
  • PCD is believed to be involved in many -neurologiGal-disorder-Sj-although-the-exact-mechanism-is-not-understood.-At-present,- early PCD can be detected only in postmortem tissue samples.
  • Reporter conjugates can be used to detect neurons that undergo PCD as follows. First, we have shown that the reporter conjugate MION-s-ODN is taken up and distributed in live neurons (see, e.g., FIGs. 2, 4, and 13). Second, once individual neurons are committed for PCD, they will either fail to take up the reporter conjugate or those that take up the reporter conjugate will be metabolized and not visible in MR or other imaging modalities. Thus, disrupted MR images of CA neurons will appear as an indication that PCD has occurred, e.g., after stroke or other neurological disorder such as Alzheimer disease or Parkinson's disease.
  • the new reporter conjugates can be used more generally for non-invasive detection of gene expression, cell mapping, gene targeting, phenotyping, and detection of gene arrays using several unique ODNs linked to different unique reporter groups (see, e.g., Example 11).
  • the new conjugates can also be used to deliver chimeric reporter groups, e.g., two or more different reporter groups linked to the same targeting nucleic acid, to specific cells, with or without the use of antibodies that specifically bind to cell-surface antigens.
  • the new reporter conjugates can be used to detect expression of an oncogene, e.g., a mutant proto-oncogene or a mutant tumor suppressor, in a tumor or cancerous cell at a very early stage in tumor development.
  • an oncogene e.g., a mutant proto-oncogene or a mutant tumor suppressor
  • Several oncogenes and tumor suppressors, such as ras and p53, are known in the art.
  • the new reporter conjugates can be used to detect the gene expression of stem cells. Stem cells have specific patterns of gene expression, depending on the type of stem cell (see, e.g., Pain et al., J. Biol. Chem., 280:6265-8 (2005)).
  • Stem cells can be visualized, e.g., following implantation (e.g., before, during, or after stem cell therapy) in a subject.
  • the new reporter conjugates can be used to detect the expression of a transgene in a subject.
  • the new reporter conjugates can be used to localize expression of a transgene in a subject.
  • the expression of a transgene that is expressed conditionally (e.g., from a conditional promoter) or tissue specifically (e.g., from a tissue-specific promoter) can be imaged using the new reporter conjugates.
  • the new reporter conjugates made with either DNA or RNA as the targeting nucleic acid can also be used to deliver any reporter molecule to any specific cellular -nue-leie- a&id ⁇ such-as-a-gene y -in-a collection of-cellular-nucleic-acids,-such-as-a-gene- bank.
  • a phosphorothioated ODN (s-ODN) 26-mer or 15-mer labeled with biotin on the 5' end was used as the targeting nucleic acid portion of the reporter conjugate; the sequence of the 26-mer has been reported (CATCATGGTCGTGGTTTGGGC AAACC (SEQ ID NO:1); Liu et al., 1994, Ann. Neurol., 36:566-576) as has that of the 15-mer (GGCAAGCCATGTCTG (SEQ ID NO:2); Paramentier-Batteur et al., 2001, Cereb. Blood Flow Metab., 21:15-21).
  • the 26-mer s-ODN binds to target c-fos mRNA and interferes with the expression of Fos/AP-1 activities after stroke of rats (Cui et al., 1999, J. Neurosci., 19:2784-2893; Zhang et al., 1999, Brain Res., 832:112-117).
  • the reporter group was a dextran-coated contrast agent, either a monocrystalline iron oxide nanoparticles (MION) or a ultra-small superparamagnetic iron oxide particle (USPIO), that was activated and conjugated using NeutrAvidin ® (Pierce Chem,).
  • Neutravidin-dextran-coated MION particles were covalently bound to the s-ODN to form the novel reporter conjugates.
  • the 3'-OH terminus of s-ODN was labeled using terminal transferase in the presence of digoxigenin (dig)-dUTP, and the resulting 5'biotin-s-ODN-3'dig was purified using a dextran column.
  • Dig-labeled 5'biotin-s-ODN was mixed with 5'biotin-s-ODN at a molar ratio of 1 :20 and stored at minus 20 0 C.
  • -NeutrAvidin— was-attaehed-to-funetional-groups-on-the-dextran-coating-on-the- MIONs and USPIOs using an aldehyde-activated dextran coupling kit (Pierce, Rockford, IL). Briefly, 20 mg of activated MION or USPIO (5 mg/ml) was added to 10 mg NeutrAvidin ® (2.5 mg/ml PBS) and the volume was adjusted using phosphate buffered saline (pH 7.4) to a final volume of 10 ml.
  • phosphate buffered saline pH 7.4
  • mice control animals with MION only and mice with the novel conjugate, MION-s-ODN (SEQ ID NO: 1).
  • Anesthesia was induced with ketamine (100 mg/kg, i.p.) plus xylazine (16 mg/kg, i.p.) to male C57bB6 mice (23-25 g, Taconic Farm, NY), and surgery was performed as described previously (Cui et al., 1999), except MION or MION-s-ODN was delivered to the brain via intracerebroventricular route (LR: -1.0, AP: -0.2, DV: -3.0 to the Bregma).
  • Example 3 MRI of Mouse Brain After the Delivery of MION-s-ODN All scanning was done in a 9.4T MRI system (Bruker-Avance). A home built
  • T2* maps 1 cm transmit/receive surface coil was placed on the head of the animal.
  • Image analysis was performed using MRVision ® software (MRVision Co, Winchester, MA), MATLAB ® (The MathWorks Inc., Natick, MA), and in-house software to construct T2* maps. In general, these acquisition sequences are readily available in any clinical MRI system. T2* maps can be calculated by the data processing software package included in the imaging system.
  • Regions of interest were extracted (as indicated in the result section), in particular along the cortices of the brain, close to as well as away from the ventricle and the injection sites.
  • T2 weighted GE images of TE 2.3 ms were compared at two time points (at less than 30 minutes, and either at 3 hours (to look for wash out) or one day (to look for retention) after infusion).
  • T2* values inversely correspond to the concentration of MION within the brain region. The orientation of presentation was from left to right, and is from the posterior to anterior part of the brain.
  • FIG. 7 provides a comparison of T2* maps of an animal that received MION-s- ODN (SEQ ID NO: 1) at ⁇ 30 minutes and one day post infusion. The expansion of reduced T2* values away from the ventricle at 1 day is indicative of MION presence.
  • FIGs. 1OA to 1OC are bar graphs (1OA and 10B) and a series of MR images (10C).
  • FIGs. 1OA and 1OB compare R2* (1/T2*) values in contralateral cortical regions (boxes in FIG. HC) from selected brain slices of mice injected with MION- sODN (SEQ ID NO:1) and MION-dextran immediately ( ⁇ 30 minutes, 10A) or 1 day after infusion (10B).
  • the bar graphs show that the brain cells retained significant amounts of the MION reporter conjugate, and that it was distributed from the left ventricle (infusion site) to the right ventricle and the cortex.
  • MION-sODN SEQ ID NO: 1
  • MION- dextran MION- dextran infusion
  • the animals were anesthetized for transcardial perfusion with 20 ml heparinized saline (2 units) at the rate of 10 ml/min, followed by 20 ml of 4% paraformaldehyde (PFA) in 0.1 M phosphate buffer (PBS), pH 7.4 at a rate of 10 ml/min ⁇ ⁇ he43rain-was-removed-and-kept4n4he-same-per-fusate-for-at-least ⁇ hours-at4- 0 C, followed by chase and storage in PBS with 20% sucrose solution.
  • PFA paraformaldehyde
  • Digoxigenin-s-ODN (SEQ ID NO:1) was stained using FITC conjugated sheep anti-dig IgG (Cui et al., 1999). The stained slides were evaluated using a fluorescence microscope to observe the presence of nuclear s-ODN. The s-ODN has been shown to be able to enter brain cells by itself when delivered via ICV route (Liu et al., 1994; Cui et al., 1999). FIGs. 4 and 13A-C show the nuclear uptake of s-ODN as clustered bright dots.
  • Example 8 Detection of Intracellular Presence of MION The presence of iron oxide was detected using Prussian blue, followed by fast nuclear red counter staining (Fisher Chem. Co).
  • iron oxide blue-green color using Prussian blue staining for iron
  • nuclear fast red nuclear fast red for nuclei counter-stain (pink-red) in animal brains that received MION-s-ODN (SEQ ID NO:1; FIGs. 3A-C).
  • Iron oxide was present in the cortex (FIG. 3B), CA neurons (FIG. 3C), and densely in the corpus callosum (FIG. 3A).
  • FIG. 3B We observed what appeared to be the diffusion of MION-s-ODN from one aggregate in the cortex (FIG. 3B). Iron oxide was also present on the molecular layer and Purkinje cells. No iron oxide was present in animals that received only MION- dextran. In addition, the presence of iron oxide in tunnels connecting cerebroventricular walls (arrows, FIG. 3A) suggests that the MIONs probably gained access to, or were removed from, brain cells via tunnels connecting the cerebro ⁇ ventricular wall for access to the CSF.
  • A26 (SEQ ID NO:1) binds specifically to c-fos -ml ⁇ A-after-eellular-uptake- ⁇ f-M-I ⁇ N ⁇ form a hybrid with c-fos mRNA in vivo and serve as a primer to enable reverse transcription (RT) towards the untranscribed region on the 5 '-terminus for a complementary DNA (cDNA).
  • RT reverse transcription
  • cDNA complementary DNA
  • Reverse transcription depends on the availability of c- fos mRNA and A26-priming, without addition of the conventional (dT) 15 as the RT primer.
  • the resulting cDNA can facilitate a subsequent amplification of itself by polymerase chain reaction (PCR) using additional c-fos specific primers.
  • the control for this experiment was MION conjugates of s-ODN with a randomized (Ran) sequence (GGGATCGTTCAGAGTCTA (SEQ ID NO: 3); MION-Ran) (Zhang et al., J. Nucl. Med., 42:1660-9 (2001)).
  • Frozen brain samples were prepared as 20 ⁇ m tissue sections and stored at - 80 0 C. All procedures were carried out in a RNase-free environment using RNase
  • Brain samples were pre-heated on a hot plate at 95 0 C following addition of PCR reaction mix (Tris-HCl [20 mM, pH 8.4], KCl [50 mM], 300 ⁇ M each of a pair of upstream and Al 8 primers, MgC12 [1.5 mM], dNTP [20 ⁇ M], dig-dUTP [1 ⁇ M] and Taq DNA polymerase [1 U, Invitrogen LT]), the reaction chamber was immediately sealed using AmpliCoverTM and clamp (Applied Biosystems, CA).
  • the PCR primers are the upstream primer which has a sequence matching to position 151 to 168 (5'- gcaactgagaagccaaga-3' (SEQ ID NO:4)), and the sequence of downstream primer Al 8 which is complementary to positions 276 - 294 (5'-catcatggtcgtggtttg-3 ' (SEQ ID NO:5)) of the c-fos mRNA (van Straaten et al., Proc. Natl. Acad. Sci.
  • thermocycler GeneAmpTM In Situ PCR system 1000, Applied Biosynthesis
  • the amplified double-stranded dig-cDNA was detected using alkaline phosphatase-anti-dig IgG and BCIP/NBT staining (Biomeda Corp, CA), or FITC-IgG against dig (Roche Applied Science, Germany) and observed directly using a mercury light source and a filter with 495 nm broad spectrum for 470 (excitation) and 525 (emission) nm wavelength). Replacement of DNase I with RNase A before reverse transcription nullified amplification.
  • RT-PCR amplification of c-fos mRNA was observed in brain samples infused with MION- A26 (FIGs. 17A and 17C), but no amplification was observed in the control in animals that received MION-Ran infusion (FIGs. 17B and 17D). The amplification was mostly observed in the cytoplasm surrounding the nucleus (asterisks) of the cortex (FIG. 17A). Moreover, no amplification was observed when c-fos primers were not present (FIG. 17E), or replaced with those of ⁇ -actin or Ran during PCR.
  • the negative control contained auto-fluorescent signal that is often associated with non- neural cells located on ventricular or vascular walls (asterisks, FIG. 17E). Therefore, the PCR signal obtained was specific for c-fos mRNA.
  • FbIR forebrain ischemia-reperfusion
  • BCCAO transient bilateral common carotid artery occlusion
  • the signals were allowed to reach steady state over a period of two days (FIG. 18A).
  • FIG. 18B shows that animals with BCCAO had significantly higher mean R2* values in the ROI of those animals that received MION- A26, but induced no changes when MION-A26 was replaced with MION-Ran (FIG. 18B).
  • A26-mediated enhancement in MR contrast was positively associated with c-fos mRNA level.
  • FbIR produced no increase in R2* values when using MION-Ran, providing strong evidence that the observed changes in R2* values of animals receiving MION- A26 were not an effect of elevated uptake of MION-s- ODN during cerebral ischemia.
  • cRNA complementary RNA
  • c-fos mRNA was slightly elevated within 30 minutes of reperfusion (release of BCCAO, FIG. 19D) and further elevated for another three hours (FIG. 19E).
  • the c-fos mRNA expression map after BCCAO showed that the highest level (in descending order) of c-fos mRNA occurred in the hippocampus (most likely the dentate gyrus (DG)), the cortex (including the SSC), and hypothalamus (HT).
  • DG dentate gyrus
  • HT hypothalamus
  • the elevation of MR contrast in the contralateral ROI shown by MR microscopy was consistent with the c- fos mRNA maps of four reperfusion times (FIGs. 19D - 19G).
  • a housekeeping gene transcript (e.g. beta actin gene, see FIG. 20B) was expressed at a higher level than c-fos mRNA (see FIG. 19C) in the brain under normal physiological condition (Cui et al., J. Neurochem., 73:1164-1174, 1999). This housekeeping gene is expressed at high level but is inert to chang by experimental or surgical protocols.
  • the MRI map in vivo also show higher R2 values for MION-s- BA25A1 (ACGCAGCTCAGTAACAGTCCGCCTA; SEQ ID NO:6) than that for c-f -os-mR ⁇ A ⁇ (seere ⁇ g ⁇ 7 -FIG. ⁇ 0G)r ⁇ he-beta-aetin-mRNA-are-expressed-in-the-c ⁇ rtex-and- hypothalamus surrounding the 4* ventricle (see, e.g., FIG. 20B).
  • An ex vivo MR map using 14 Tesla system also show elevated R2* map in the cortex and the hypothalamus (see, e.g., FIG.
  • MR images were acquired in live animals (with pure O 2 plus 2% halothane [800 ml/min flow rate]) using a 9.4 Tesla magnet.
  • FIG. 21 A shows R2* values (mean and SE) in the contra-lateral hippocampus of 5 animals 7 days after a global stroke.
  • Nissl and TUNEL staining showed nuclear and DNA fragmentation consistent with apoptosis. These data are consistent with the hypothesis that apoptotic neurons do not retain MION-ODN. The absence of MION-retention in hippocampal neurons after stroke indicated

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Abstract

L'invention concerne un conjugué rapporteur destiné à l'imagerie non invasive de l'expression génique in vivo. Le conjugué comporte un acide nucléique de ciblage lié à un agent de contraste, par exemple une étiquette paramagnétique que l'on peut utiliser avec l'imagerie par résonance magnétique (IRM). L'acide nucléique de ciblage peut être un brin antisens qui s'hybride en une partie d'un ARN messager codé par le gène dont l'expression doit être imagée. Dans certains modes de réalisation, l'agent de contraste est un métal chélaté tel que le gadolinium ou le dysprosium. L'invention concerne également des procédés permettant d'imager une expression génique dans différents tissus, y compris le cerveau.
PCT/US2005/029875 2004-08-23 2005-08-23 Imagerie d'acides nucleiques cellulaires WO2006023888A2 (fr)

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WO2007141305A2 (fr) 2006-06-06 2007-12-13 Guerbet L' imagerie de diffusion de l' eau et uspio
EP1865839A4 (fr) * 2005-03-21 2011-06-29 Univ California Nanoparticules magnetiques fonctionnalisees et leurs methodes d'utilisation

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US651967A (en) * 1897-11-12 1900-06-19 Peter J Freize Chainless-wheel bicycle.

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1865839A4 (fr) * 2005-03-21 2011-06-29 Univ California Nanoparticules magnetiques fonctionnalisees et leurs methodes d'utilisation
WO2007141305A2 (fr) 2006-06-06 2007-12-13 Guerbet L' imagerie de diffusion de l' eau et uspio
FR2918868A1 (fr) 2006-06-06 2009-01-23 Guerbet Sa Methode d'imagerie de diagnostic utilisant en combinaison avec l'imagerie de diffusion de l'eau, des agents de contraste

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