WO1999061071A2 - Techniques permettant de marquer des vecteurs a base d'acides nucleiques avec des oligonucleotides formant des triplex et d'analyser la distribution desdits vecteurs - Google Patents

Techniques permettant de marquer des vecteurs a base d'acides nucleiques avec des oligonucleotides formant des triplex et d'analyser la distribution desdits vecteurs Download PDF

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WO1999061071A2
WO1999061071A2 PCT/US1999/011511 US9911511W WO9961071A2 WO 1999061071 A2 WO1999061071 A2 WO 1999061071A2 US 9911511 W US9911511 W US 9911511W WO 9961071 A2 WO9961071 A2 WO 9961071A2
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tfo
nucleic acid
triplex
labeled
triplexes
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PCT/US1999/011511
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WO1999061071A3 (fr
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Igor G. Panyutin
Ronald D. Neumann
Andrew N. Luu
Olga A. Sedelnikova
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The Government Of The United States Of America, Represented By The Secretary, Department Of Health Aand Human Services
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Priority to AU43124/99A priority Critical patent/AU4312499A/en
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Publication of WO1999061071A3 publication Critical patent/WO1999061071A3/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
    • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
    • A61K51/04—Organic compounds
    • A61K51/0491—Sugars, nucleosides, nucleotides, oligonucleotides, nucleic acids, e.g. DNA, RNA, nucleic acid aptamers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6839—Triple helix formation or other higher order conformations in hybridisation assays

Definitions

  • the present invention pertains to methods for labeling nucleic acids such as recombinant double-stranded DNA (dsDNA) expression vectors and, following a(irninistrauon of the labeled nucleic acid molecules to a living animal or human, non- invasively measuring the distribution of the labeled nucleic acid molecules in organs and tissues of the subject's body.
  • the present invention provides a rapid, non-invasive means for assessing the effectiveness of techniques for delivering therapeutic gene expression vectors to their target tissues in vivo.
  • the present invention also pertains to methods in which nucleic acids such as dsDNA molecules are labeled and are introduced into cells, and the intracellular disposition of the labeled DNA molecules is determined in vitro.
  • triplex ⁇ brming oligonucleotides (TFOs) labeled with detectable chemical groups are allowed to bind to native dsDNA molecules to form triple-stranded nucleic acid complexes (triplexes) that are stable inside of cells under normal growth conditions, (ii) the resulting labeled triplex-contairiing DNA molecules are introduced into a multicellular organism or into cultured cells, and (iii) the distribution of the labeled DNA molecules in the treated organism or cells is quantitatively monitored by detecting the labeled triplex DNA molecules in the subject organism or cells.
  • Non-invasive imaging methods such as
  • MRI Nuclear Magnetic Resonance Imaging
  • PET Positron Emission Tomography
  • Intracellular distribution of the labeled DNAs is carried out by commonly used methods for detecting labeled molecules in cells; e.g., by visual detection of fluorescent labels fluorescence microscopy, or by detection of radiolabels by autoradiography.
  • Efforts to treat disease by administering recombinant nucleic acid expression vectors carrying therapeutic genes as pharmaceutical agents (gene therapy) to human patients who would benefit from expression of the genes in specific cells and tissues of their bodies are generally confounded by the inability of currently available methods to deliver therapeutically effective amounts of the nucleic acid expression vectors to target cells and tissues of the patients.
  • Biodistribution of DNA expression vectors carrying the firefly luciferase reporter gene has been monitored non-invasively in mice and zebrafish by detecting biolumincsccnce in tissues where the luciferase gene is expressed (Contag et al., Photochem. Photobiology, 1997, 66(4):523-31; Patil et al., Zoological Science, 1994, 11 (l):63-68); however, it is unclear whether bioluminescence can be detected non-invasivcly in internal tissues of large animals such as humans.
  • DNA oligonucleotides and their analogs conjugated to imaging agents such as fluorine-18 (" ⁇ ), technetium-99m CTc) and indium-I l l ('"-In) have been administered to experimental animals, and their biodistribution in the animals has been monitored by PET (Tavitian et al., Nature Medicine, 1998, 4(4):467-71) and whole-body gamma camera imaging (Dewanjee et al., The Journal of Nuclear Medicine, 1994, 35(6):1054-63; Hnatowich et al., Journal of Pharmacology and Experimental Therapeutics, 1996, 276(l):326-34).
  • TFOs triplex-forming oligonucleotides
  • Pyrimidine motif TFOs are composed of pyrimidines comprising thymine (T) and cytosine (C), and bind in parallel orientation to a run of purines in duplex DNA by Hoogsteen base-pairing in the major groove of the DNA, with T in the TFO pairing with adenine (A) in the target DNA, and C in the TFO pairing with guanine (G) in the target DNA.
  • Purine motif nucleotides are composed of purines comprising A and G, and bind to a run of purines in duplex DNA by reverse Hoogsteen base- pairing in the major groove, with G in the TFO pairing with G in the target DNA, and A in the TFO pairing with A in the target DNA (Beal et al., Science, 1991, 251:1360-3; Debin et al., Nucleic Acids Research, 1997, 25(10):1965-74; Dervan et al., U.S. Patent No. 5,874,555, in entirety).
  • GT-type TFOs comprise G and T bases, and bind in the major groove to a run of at least about 65% purines in duplex DNA, with G in the TFO opposite a GC pair in the target DNA, and T in the TFO opposite an AT pair in the target DNA; the binding may be in either parallel or anti-parallel orientation, depending on the target nucleeotide sequence (Hogan et al., U.S. Patent No. 5,176,996, in entirety; Debin et al., Nucleic Acids Research, 1997, 25(10):1965). Debin et al.
  • the present invention provides methods in which TFOs are labeled with chemical moieties that are readily detected by imaging systems, the labeled TFOs are bound to target sequences in nucleic acid molecules in vitro to form triplex complexes, the labeled triplex complexes are introduced into a living organism, and non-invasive imaging means are used to quantitatively monitor the biodistribution of the labeled nucleic acid molecules in the organism.
  • the nucleic acid molecules of the present method are selected to have the same size and structure as nucleic acid gene expression vectors, e.g., dsDNA plasmids, that are designed for gene therapy.
  • the TFOs are labeled with detectable chemical groups, for example, with paramagnetic metal ions detectable by MRI, with gamma- or positron- emitting radionuclides detectable by a gamma camera or PET, or with a lluorophore detectable by its fluorescence in ultraviolet light.
  • the labeled TFOs are complexed with nucleic acids molecules to form triplex complexes, and the triplexes are administered to a living organism.
  • the biodistribution of the triplex nucleic acid molecules in the subject organism after one or more time intervals subsequent to administration of the triplexes is then monitored by a device that detects the labeled TFOs.
  • the present invention permits monitoring the intracellular distribution of nucleic acid vectors such as dsDNA plasmids by allowing the nucleic acid vectors to form triplex complexes with TFOs that are labeled with a fluorophore, introducing the labled triplexes into cells, and viewing the cells in vitro using fluorescent and confocal microscopy.
  • Figure 1 is a map of the pCR3HPRT plasmid.
  • the 833 base pair (bp) PCR fragment cloned into pCR3 vector (pos. 724-1556) is shown in bold.
  • the amplified inset shows the target sequence along with TFO. Positions of '"I dC's are marked with stars.
  • the broad line between the Hind III and Nde sites represents the fragment cut from the plasmid by Hind III and Nde I restriction enzymes for use as probe for Southern hybridization.
  • Figure 2 shows the rate of triplex formation in vitro (Figs. 2 A and 2B) and the dependence of the rate of binding on plasmid concentration (Figs. 2C and 2D).
  • Figs. 2A and 2B 1 nM 125 I-TFO and 10 nM pCR3HPRT plasmid were incubated at 37°C for 0, 1, 5, and 24 hrs. in TMSp buffer, the DNA molecules were electrophoresed in 2% agarose, the distribution of radioactivity in the gel was measured
  • Figs. 2C and 2D different concentrations of plasmid (0-50 nM) were incubated with a constant concentration of 125 I-TFO overnight, the DNA molecules were electrophoresed in 2% agarose, the distribution of radioactivity in the gel was measured
  • Figure 3 shows the dependence of triplex stability in vitro on temperature
  • Figure 3 A pre-formed triplex was diluted in a buffer containing 0.5 mM MgC and aliquots were incubated at room temperature, 37°C, and 50°C for 30 min, and at
  • Figure 3B pre-formed triplex was diluted in buffer to ImM Mg *2 and aliquots were incubated for 10 min at 37°C with 0.5, 1.0, 2.5, and 5.0 mM EDTA, which chelates Mg *2 , the DNA molecules were electrophoresed in 2% agarose, and the distribution of radioactivity ( 12, I-TFO) in the gel was measured. Sixty percent of the triplexes incubated with 0.5 mM EDTA dissociated, and all the triplexes incubated with higher concentrations of EDTA dissociated completely.
  • Figure 4 shows that the number of breaks introduced by 1 T-TFO in dsDNA in vitro is directly proportional to the fraction of plasmid to which 12 T-TFO is bound. Triplexes were formed in the presence of different concentrations of 12i I-TFO, and then were frozen and incubated at -70°C to permit strand breaks to occur.
  • Figure 4A shows the distribution of ethidium bromide-labeled DNA molecules in an agarose gel following electrophoresis. The percentage of the plasmid carrying TFO is shown at the top.
  • Figure 4B shows an autoradiograph made following Southern blot of the gel, and hybridization with a radio-labeled probe.
  • Figure 4C shows the percentage of DNA breaks plotted against the percentage of plasmid bound to TFO. Percent of breaks was calculated as the ratio of the sum of the intensities of the two shorter bands in the case of ethidium stained gel (•) and the intensity of the single shorter band in the case of Southern blot ( ⁇ ) to the total intensities of the bands in lanes.
  • Figure 5 (A &B): Triplex radioprinting in cells.
  • Fig. 5A Southern blot of Hirt extracts from HeLa cells that have been transformed with preformed triplexes ( 12 T-TFO/pCR3HPRT). Cells were incubated with triplexes for 5 hr, postincubated for 17-43 hr, then frozen and stored at -70°C to accumulate 12S I decays. The recovered plasmid was cut with Pvul, and the fragments were separated by electrophoresis and transferred to a hybridization blot. The blot was hybridized with 32 P-labeled Hind III-Nde I probe. The positive control was triplex that was stored in a test tube without delivery into cells (Triplex). The negative control was pCR3HPRT plasmid delivered into cells and Hirt-extracted (Plasmid).
  • Fig. 5B Percent double-strand plasmid DNA breaks vs. time of incubation of the cells with preformed triplexes.
  • Figure 6 (A-D) shows the distribution of triplexes inside cells.
  • HeLa cells were transfected with pre-formed triplexes (FITC-TFO/pCR3HPRT plasmid) complexed with DMRIE liposomes for 5 hr.
  • Confocal ( Figure 6A) and fluorescent ( Figure 6C) microscope analysis showed that FITC-labeled triplexes were equally distributed in cytoplasm and nuclei of transfected cells.
  • Confocal ( Figure 6B) and fluorescent ( Figure 6D) microscopy of control cells transfected for 5 hr with FITC- TFO/DMRIE shows that fluorescence is concentrated in bright grains in cytoplasm. Magnification: lOOx.
  • Figure 7 compares assay of plasmid uptake based on physical detection of triplexes to assay based on detecting reporter gene expression.
  • Fig. 7A shows HeLa cells transfected with pCMV-sport- ⁇ -gal plasmid, stained with ⁇ -gal staining kit, and counterstained with nuclear fast red.
  • Fig. 7B shows HeLa cells transfected with preformed FITC-TFO/ pCR3HPRT triplexes and monitored for flourescence.
  • Fig. 7C shows HeLa calls transfected with preformed 125 I-TFO/pCR3HPRT triplexes (autoradiography); and counterstained with hematoxylin and eosin. Magnification: 40x.
  • the method of the present invention operates with any nucleic acid molecule that contains a target nucleotide sequence to which a labeled TFO stably binds to form a triplex complex.
  • the nucleic acid molecules which bind the labeled TFOs to form triplexes can be single- or double-stranded DNA or RNA molecules that are linear or circular, native or recombinant, and they can be plasmids, viral genomes, episomes, or artificial chromosomes (see Huxley, Gene Therapy, 1994, 1(1):7-12).
  • the TFO-binding nucleic acids of the present invention are circular dsDNA molecules such as plasmids.
  • DNA and RNA oligonucleotides having a number of different structures function effectively as TFOs to form stable triplex complexes with specific sequences in target DNA and RNA molecules.
  • TFOs DNA and RNA TFOs that bind to single-stranded DNA and RNA molecules, such as fold- back TFOs (Hiratou et al., Nucleic Acids Symposium Series, 1997, 37:221-2; Kandimalla et al., Nucleic Acids Research, 1995, 23 (6): 1068-74), tethered TFOs (Moses et al., Bioorganic Med. Chemistry, 1997, 5(6):1123-9), dsDNA probes
  • TFOs that are structurally modified to have increased triplex stability or resistance to nucleases; e.g., by introducing nucleoside analogs (Wang et al., Bioorganic
  • the TFOs of the present invention are single-stranded, polypurine, deoxynucleotide TFOs.
  • stably bound means that the labeled TFO remains bound to the dsDNA under physiological conditions for a time period of sufficient duration that monitoring of biodistribution can be carried out.
  • the time period of stability of the triplex required for monitoring biodistribution be from 10 minutes or less to about one hour, or to as long as two or more days, depending on the goal and design of the assay protocol.
  • physiological conditions refers to chemical and physical conditions in cells or in tissues in a living organism in which dsDNA biodistribution is to be monitored.
  • the detectable labeling agents of the present invention are compounds that arc detected in vivo by accepted non-invasive techniques in the art of diagnostic imaging.
  • radionuclide atoms can be used to label detectably the TFOs in accordance with the present invention. These include both gamma-emitters and positron emitters; examples of which include, but are not limited to, fluorine-18, copper-64, copper-65, gallium-67, gallium-68, bromine-77, ruthenium-95, ruthenium- 97, ruthenium- 103, ruthenium- 105, technetium-99m, mercury 107, mercury-203, iodine-123, iodine-125, iodine-126, iodine-131, iodine-133, indium-I l l, indium-113m, rhenium-99m, rhenium-105, rhenium-101, rhenium-186, rhenium-188, tellurium- 121m, telurium-122m, tellurium-125m, thulium-165,
  • Metal atoms detectable by MRI such as ions of manganese, iron, gadolinium, terbium, dysprosium, holmium, erbium, ytterbium, lutetium, holmium, bismuth, lead and hafnium, can also be used as labeling agents.
  • metal ions such as those listed above can be bound by chelating moieties, which in turn can be conjugated to the TFOs of the present invention.
  • gadolinium ions are chelated by diethylenetriaminepentaacetic acid (DTP A), and a number of lanthanide ions, including gadolinium and dysprosium, are chelated by terraazacyclododocane compounds (Klaveness et al., U.S. Patent No. 5,738,837, in entirety; and Meade et al., U.S. Patent No.
  • Tc is chelated by an N-hydroxysuccinimide derivative of mercaptoacetyltriglycine (NHS-MAGs, see Mardirossian et al., The Journal of Nuclear Medicine, 1997, 38(6):908); and '"In is chelated by DTPA isothiocyanate (Dewanjee et al., The Journal of Nuclear Medicine, 1994, 35(6):1055).
  • the chelating groups can be attached to the TFOs by known methods, e.g., via a nitrogen atom introduced at one of the TFO termini (as in Dewanjee et al., The
  • ATFO that is detectable by PET can be produced, for example, by attaching a chemical moiety containing a positron- emitting 18 F atom to either TFO terminus (Tavitian et al., Nature Medicine, 1998, 4(4):467-71), or by incorporating a nucleoside analog containing a positron-emitting "C atom into the TFO (Conti et al., 1995, Nucl. Med. Biol. 22(6):783-789).
  • Labeled TFOs are incubated in the presence of the target nucleic acids and divalent cations to obtain formation of stable triplex complexes using routine methods known by those skilled in the art.
  • pharmaceutically acceptable means acceptable for use in the pharmaceutical and veterinary arts; i.e., a carrier which is non-toxic and which does not adversely affect the activity of the composition in its function to deliver the labeled nucleic acids to their target tissues, or the monitoring of the biodistribution of the labeled nucleic acids. It is within the knowledge of those skilled in the art of compositions comprising nucleic acid vectors for administration in vivo to select and include a pharmaceutically acceptable carrier in the compositions of the present invention.
  • compositions comprising nucleic acid vectors for administration in vivo recognize drat such compositions can also comprise chemical agents that assist in delivering the nucleic acids into their targeted cells in vivo, such as anionic or cationic lipids, polycations, and compounds that bind to specific cell-surface receptors and promote introduction of the nucleic acids into the cells bearing the receptors on their surface (see, for example, Alino, Biochem. Pharmacology, 1997, 54(1):9-13; Liu et al., Journal of Biological Chemistry, 1995, 270(42) :24864-70; Hong et al., FEBS Letters, 1997, 400(2):233-7; Thierry et al.,
  • the labeling moiety attached to the TFO is a radionuclide
  • stabilizers to prevent or minimize radiolytic damage such as ascorbic acid, gentisic acid, or other appropriate antioxidants, may be added to the composition comprising die labeled triplexes that is administered to the subject organism.
  • compositions comprising labeled triplex complexes for use in monitoring biodistribution of the triplexes in a subject animal in accordance with the present invention can be administered by many of the same routes that are commonly used to administer conventional drugs; for example, by intravenous, intraperitoneal, or intramuscular injection, by aerosol inhalation, by intratracheal installation, and by injection direcdy into a target tissue (e.g., a tumor) (for example, sec Canonico et al., Journal of Applied Physiology, 1994, 77(l):415-9; Cooper, Seminars in Oncology,
  • compositions comprising detectably labeled triplexes of the present invention are established in controlled trials, and correspond to an amount sufficient to allow detection of the labeled triplexes in tissues of the subject organism following administration, as compared to the background signal obtained upon administration of an appropriate control composition, without causing intolerable side effects and without unacceptable exposure to radioactivity.
  • the dosages required to obtain a desired measure of biodistribution will vary according to the specific organism or individual used as the subject (i.e., species, age, sex, and general health), the chemical make-up of the triplex-containing composition (liposomes, cationic lipids, polyanions, targeting peptides, etc.), the route of administration, the type of labeling moiety, and the imaging method (MRI, PET, SPECT, etc.) that are used.
  • die instrumentation that may be used in the detection of die labeled TFOs depends on the type of label attached to the triplexes, and on the type of target tissue or cells being imaged.
  • a superconducting quantum interference device magnetometer SQUID, see Klaveness et al., U.S. Patent No. 5,738,837.
  • a gamma camera and a rectilinear scanner each represent instruments useful to detect radioactivity in a single plane.
  • Single Photon Emission Computed Tomography (SPECT) and PET devices represent instruments that are capable of detecting radioactivity in more than one dimension.
  • SPECT Single Photon Emission Computed Tomography
  • PET devices represent instruments that are capable of detecting radioactivity in more than one dimension.
  • Imaging instruments suitable for practicing the methods of the present invention are readily available from commercial sources in the U.S. (for example, for PET: ADAC, Milpitas, CA; Siemens, Hoffman Estates, IL; Concorde Microsystems, Inc, Knoxville, TN; for MRI: Picker International, Inc., Cleveland, OH; Siemens, Iselin, NJ; GE, Waukesha, WI; and for SPECT: Toshiba America/USA, Tustin, CA; Siemens, Hoffman Estates, IL; ADAC,
  • Radioprinting a method for detecting triplex complexes comprising '"I-labeled TFOs based on measurement of DNA strand breaks at sites in the target duplex DNA proximal to the decay site (Panyutin et al., Nucleic Acids Research, 1994, 22(23):4979-82; Panyutin et al., Nucleic Acids Research, 1997, 25(4):883-7). Radioprinting may be used to determine the stability of a triplex complex comprising a TFO and a nucleic acid of interest under physiological conditions.
  • Nucleic acid to be labeled pCR3HPRT plasmid containing the triple helix-forming polypurine- polypyrimidine region of human hypoxanthine-guanine phosphoribosyl transferase (HPRT) gene was constructed by inserting the PCR-amplified fragment into pCR3 vector (Invitrogene, Carlsbad, CA) (Panyutin et al., Nucleic Acids Research, 1994, 22(23):4979-82).
  • a map of the pCR3HPRT plasmid containing the 832 bp insert from the human HPRT gene intron A (Panyutin et al., Acta Oncol., 1996, 36:817-824;
  • Fig.l Prior to use, supercoiled plasmid was relaxed with topoisomerase I (Promega, Madison, WI).
  • pCMV-sport- ⁇ - gal plasmid containing ⁇ -galactosidase gene was purchased from Gibco BRL. All plasmids were purified by centrifugation through CsCl gradient.
  • Oligonucleotides were synthesized on an ABI-394 DNA syndiesizer (Applied Biosystems, Foster City, CA) followed by purification from a polyacrylamide gel (PAG).
  • the template oligonucleotide was biotinylated using BioTEG modifiers (Glen
  • Phosphodiester TFOs were labeled with 12 -dCTP at the C5 position of three cytosines (marked with stars on Fig. 1) by primer extension method (Panyutin et al., Acta Oncol., 1996, 36:817-824; Panyutin et al., Nucleic Acids Research, 1997, 25(4):883-7).
  • the product TFOs were estimated to have 1.5 1M I per oligonucleotide.
  • Fluorescein (FITC) labeled TFOs were synthesized using FITC modifiers (Glen Research). Triplex formation and stability
  • TMSp buffer containing 50 mM Tris-HCl, pH 8, 10 mM MgCL, 0.1 mM Spermidine and 16 ⁇ M Coralyne (15-17).
  • Aliquots of the samples were analyzed by 2% Agarose gel at 10°C using TAE buffer containing 3 mM MgAc as an electrode buffer for 1 hr at 60 volts. Gels were analyzed with a BAS 1500 Bio-Imaging Analyzer (Fuji, Tokyo, Japan) or Fluo-Imager (Molecular Dynamics, Sunnyvale, CA).
  • the human cervical epithelial carcinoma HeLa cell line (ATCC, Manassas,
  • VA CCL2
  • DMEM Dulbecco's modified Eagle's minimum essential medium
  • FBS fetal bovine serum
  • DMRIE liposomes (Gibco BRL, Gaithersburg, MD) were used. Five ⁇ l of DMRIE (initial concentration 2mg/ml) was diluted in 45 ⁇ l OptiMEM medium (Gibco BRL, Gaithersburg, MD). Three ⁇ g of plasmid DNA (or plasmid/TFO triplex) was diluted in OptiMEM to 50 ⁇ l. Fifty ⁇ l of the liposomes solution were mixed with DNA solution, and the mixture was left for 20- 30 min at room temperature before further use.
  • OptiMEM medium 900 ⁇ l of OptiMEM was added to each well and the cells were put in 37°C CO a incubator until further use. DNA/DMRIE complexes (100 ⁇ l) were added to each well. The cells were incubated for 5 hrs, washed with DMEM containing 3 mM EDTA, and then postincubated in DMEM medium for die desired time. Alter the incubation was completed, the cells were trypsinized, collected, frozen, and stored at -70°C to accumulate ""I decays.
  • the transfected cells were fixed in fixative solution (2% formaldehyde, 0.2% glutaraldehyde in PBS), and stained with ⁇ -gal staining kit (Invitrogen). The amount of blue cells was counted with a microscope (Karl Zeiss, Oberkochen, Germany). To check the viability of transfected cells, cells were stained with trypan blue and counted the number of dead and survived cells with a microscope. The cells were then analyzed with fluorescent and confocal microscopy, and by autoradiography (Sedelnikova et al., Journal of Nuclear Medicine, 1998, 39:1412-1418).
  • RNA was extracted twice with equal volumes of phenol/chloroform, and then once with chloroform.
  • the supernatant from the last extraction was placed into a microtube containing 150 ⁇ l of 10 M ammonium acetate and 700 ⁇ l of isopropanol. The tube was inverted several times and spun for 30 min in microcentrifuge. The pellet was dried and resuspended in 50 ⁇ l of TE buffer, then precipitated with ethanol, washed with 70% cold ethanol, and spun again. The dried pellet was resuspended in TE buffer.
  • Plasmid DNA was electrophoresed through 1% agarose gel and transferred to a Genescreen hybridization membrane (NEN Life Science Products, Boston, MA).
  • the 405 base pair Hindlll/Ndel fragment of pCR3HPRT plasmid was labeled with 32 P using an oligolabeling kit (Pharmacia Biotech, Piscataway, NJ), and the 32 P labeled oliginucleotides were incubated with the hybridization membrane, following die manulacturer's instructions, and then were visualized with a BAS 1500 Bio-Imaging Analyser. Kinetics of triplex formation
  • Plasmid (10 nM) was incubated with ra ⁇ -TFO (1 nM) at 37°C for 1 hr, 5 hr and 24 hr in TMSp buffer. As a control, an aliquot of the mixture was not incubated and kept on ice (0 hrs). Formation of triplex complexes was monitored by gel shift assay (Panyutin et al., Nucleic Acids Research, 1994, 22(23) :4979-82; Panyutin et al., Nucleic
  • triplex stability in vitro The triplex complexes were found to be stable alter dilution and incubation at elevated temperatures. To determine the stability of the triplex complexes in vitro, an aliquot of the preformed triplex was diluted 10 times in 20 mM Tris-HCl, 20 mM NaCl, 0.5 mM MgCL buffer and kept overnight at room temperature. Aliquots of the diluted sample were also incubated at 37°C, 50°C for 30 min, and at 90°C for 1 min. Fig. 3A shows that the triplexes remained stable at all the conditions, and complete dissociation of the triplexes was observed only after heating at 90°C .
  • aliquots of the preformed triplex complexes were diluted in 20 mM Tris-HCl, 20 mM NaCl, buffer without MgCL (the final concentration of magnesium was ImM), and incubated with 0.5, 1, 2.5, and 5 mM EDTA for 10 min at 37°C.
  • Fig. 3B 60% of the triplexes incubated with 0.5 mM EDTA dissociated, and the triplexes incubated with higher concentrations of EDTA dissociated completely.
  • Radioprinting was first demonstrated using conventional analytical methods which could monitor triplex formation, such as the gel shift assay.
  • a series of samples with different percent of triplex formation was prepared. All the samples contained 80 nM of pCR3HPRT plasmid and the following concentrations of ,25 I-TFO; 8nM, 20nM,
  • DNA-containing bands from a gel similar to one shown in Fig. 4A were transfered to a nylon membrane and hybridized with the 32 P-labeled Hindlll-Ndel probe (Figs. 1 and 4B). The probe hybridized only with the 2.15 kb fragment. The intensity of the band corresponding to that fragment increases with increase of the percentage of the triplex.
  • Fig. 4C shows the percent of breaks, calculated from the ratio of the intensity of the decay-produced bands to the total intensity of all bands in the lanes, plotted against the percentage of triplexes. The 12ii I-TFO produced breaks are direcdy proportional to the percent of the triplexes on the plasmid.
  • quantitation of breaks introduced into a nucleic acid by bound 125 I-TFOs by the radioprinting assay provides an accurate measure of the amount of the nucleic acid that is present in triplex form.
  • the relative number of breaks in the plasmid, and therefore the fraction of the plasmid in triplex form can be quantitatively determined either by direct staining with ethidium bromide or by Southern hybridization, as shown in Fig. 4C.
  • the slighdy lower values of the percentage of breaks in the hybridization experiments as compared with the ethidium bromide sterining can be attributed to the weaker probe hybridization with the shorter fragment.
  • Plasmid DNAs were delivered into HeLa cells using DMRIE liposomes. Transfection conditions were optimized with pCMV-sport- ⁇ -gal plasmid to obtain 60% transfection efficiency, i.e. 60% of transfected cells expressed ⁇ -galactosidase gene and became blue after staining with " ⁇ -gal Staining Kit" (Fig. 7A). Staining with trypan blue showed that almost 90% cells survived transfection (not shown). When preformed 12 T- TFO/pCR3HPRT triplexes were delivered into cells, 30% of radioactivity was associated with cell pellet after 5-hr incubation.
  • the intracellular stability of triplex complexes formed from equal amounts of pCR3HPRT plasmid and 12, I-TFO was analyzed by the radioprinting assay.
  • the preformed triplex complexes were transfected into HeLa cells by incubating the cells for 5 hours with DMREI liposomes containing the triplexes, and then washing the cells with DMEM/3mM EDTA.
  • the concentration of EDTA in the wash solution is considered to be sufficient to dissociate any triplex complexes remaining outside of the cells.
  • the DMEM/EDTA wash the cells were post-incubated in DMEM for 20, 31, 38, and 48 hrs., collected, and frozen for 60 days.
  • plasmid DNA was extracted, cut with Pvul restriction enzyme and analyzed in 1% agarose gel, followed by Southern hybridization with Hindlll-Ndel probe to detect 12' I-TFO induced breaks.
  • the distribution of radioactivity in die gel is shown in Fig. 5A, with lane 1 containing the control triplex that had been frozen in a test tube for the same amount of time.
  • Fig. 5B all of the analyzed samples contained approximately the same amount of breaks; about 20%, which means that the triplexes remained stable inside the living cells for at least 48 hrs, with no significant dissociation of TFO from the plasmid during that time.
  • FITC-labeled TFOs were used to visualize the distribution of plasmid/TFO complexes inside cells.
  • HeLa cells were transfected with preformed FITC- TFO/pCR3HPRT triplexes using DMRIE liposomes.
  • the cells were transfected with FITC-TFO alone using the same liposomes.
  • Fluorescent (Figs. 6A and 6B) and confocal (Figs. 6C and 6D) microscopy showed that FITC labeled triplexes were uniformly distributed in the cytoplasm and nuclei of the cells, whereas the unbound FITC-labeled TFOs were not released from the liposomes, and remained concentrated in bright grains in the cytoplasm.
  • Fluorescent signal from the FITC-TFO/pCR3HPRT triplexes was observed in almost all of the cells; however, measurement of expression of the ⁇ -galactosidase reporter gene in the cells revealed expression of the gene in only half of the cell population (Figs. 7 A and 7B).
  • Autoradiography confirmed introduction of the 125 I- TFO/pCR3HPRT triplexes into nearly all the cells (Fig. 7C). This illustrates that physical detection of triplexes containing labeled TFOs permits quantitative measurement of the presence of the labeled triplexes that is not possible by assaying for expression of reporter genes.
  • Radioprinting assay described above allows detection of the interaction of TFOs with their target sequences both in vitro and in vivo.
  • the method is based on a unique property of Auger electron emitters, such as 'T, 1 3 I, "'In and others, to produce DNA breaks within close proximity to the decay site.
  • Radioprinting can be considered as a general form of radioprobing, a method that allows one to detect conformational changes in DNA structure by measuring distribution of breaks induced by Auger electron emitters with single nucleotide resolution (Panyutin et al., Nucleic Acids Research, 1997, 25(4):883-887).
  • the assays of triplex stability described above were performed with commercially available 12i I-dCTP and can be easily repeated in any laboratory.
  • the radioprinting methods can be extended to detect interaction of a target DNA or RNA sequence with other types of ligands labeled with Auger electron emitters, such as a proteins, non-triplex forming nucleic acids, antibiotics and other low molecular weight compounds.

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Abstract

Des oligonucléotides formant des triplex (TFO) peuvent former in vitro, avec des séquences nucléotidiques cibles contenues dans un acide nucléique, des complexes de triplex qui, après introduction dans des cellules eucaryotes en culture, restent stables à l'intérieur de celles-ci au moins 48 heures. La stabilité de ces complexes de triplex formés par liaison de TFO marqués à l'iode 125 à un acide nucléique cible peut être contrôlée quantitativement par radio-impression. Le nombre de cassures de brins d'ADN provoquées par l'iode 125 introduit dans l'acide nucléique cible est déterminé. Ce nombre est proportionnel au nombre de complexes de triplex présents dans l'acide nucléique. Selon l'invention, les TFO sont marqués in vitro au moyen d'agents d'imagerie pouvant être détectés in vivo par des techniques d'imagerie telles que l'imagerie à gamma-caméra, le scanner à balayage rectiligne, l'IRM, la tomographie à émission de positrons (PET) et la tomographie d'émission monophotonique (SPECT). On laisse les TFO marqués se lier à leur acide nucléique cible et former avec lui des complexes de triplex, puis on administre à un organisme pluricellulaire une composition pharmaceutique renfermant les triplex marqués. On utilise ensuite les techniques d'imagerie capables de détecter l'acide nucléique marqué afin de contrôler de manière non invasive la biodistribution de l'acide nucléique dans l'organisme du patient. L'invention constitue ainsi un nouvel outil important pour développer les techniques d'apport d'acides nucléiques faisant partie de protocoles de thérapie génique.
PCT/US1999/011511 1998-05-26 1999-05-26 Techniques permettant de marquer des vecteurs a base d'acides nucleiques avec des oligonucleotides formant des triplex et d'analyser la distribution desdits vecteurs WO1999061071A2 (fr)

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Cited By (2)

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Publication number Priority date Publication date Assignee Title
WO2002024901A1 (fr) * 2000-09-19 2002-03-28 Takara Bio Inc. Procede de formation de complexes
US11208677B2 (en) 2018-06-07 2021-12-28 Amgen Inc. Detection assay for protein-polynucleotide conjugates

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002024901A1 (fr) * 2000-09-19 2002-03-28 Takara Bio Inc. Procede de formation de complexes
US11208677B2 (en) 2018-06-07 2021-12-28 Amgen Inc. Detection assay for protein-polynucleotide conjugates

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