[go: up one dir, main page]

WO1991011535A1 - Inhibition de transcription a l'aide d'oligonucleotides bicatenaires - Google Patents

Inhibition de transcription a l'aide d'oligonucleotides bicatenaires Download PDF

Info

Publication number
WO1991011535A1
WO1991011535A1 PCT/US1991/000635 US9100635W WO9111535A1 WO 1991011535 A1 WO1991011535 A1 WO 1991011535A1 US 9100635 W US9100635 W US 9100635W WO 9111535 A1 WO9111535 A1 WO 9111535A1
Authority
WO
WIPO (PCT)
Prior art keywords
transcription
gene
oligonucleotides
oligonucleotide
sequence
Prior art date
Application number
PCT/US1991/000635
Other languages
English (en)
Inventor
John Holcenberg
Henry Wu
Original Assignee
Childrens Hospital Of Los Angeles
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Childrens Hospital Of Los Angeles filed Critical Childrens Hospital Of Los Angeles
Publication of WO1991011535A1 publication Critical patent/WO1991011535A1/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1131—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against viruses
    • C12N15/1132—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against viruses against retroviridae, e.g. HIV
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/67—General methods for enhancing the expression
    • 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
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • C12Q1/701—Specific hybridization probes
    • C12Q1/702—Specific hybridization probes for retroviruses
    • C12Q1/703—Viruses associated with AIDS
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/13—Decoys
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/31—Chemical structure of the backbone
    • C12N2310/315—Phosphorothioates

Definitions

  • the present invention relates to novel methods for controlling gene expression in which double stranded
  • oligonucleotides are used to inhibit the interaction of transcriptional factors with transcriptional control elements in DNA.
  • the methods of the invention are particularly useful in selectively inhibiting transcription of viral genes and oncogenes, and may be used in the
  • nucleosides are joined by phosphorothioate linkage.
  • 5 in vitro binding assays such as footprinting and gel retardation analysis, have been useful in delineating promoter function ( u, C. , 1986, Nature, 1985, 317:84-87 and Singh et al., 1986, Nature, 319: 154-158) .
  • These studies have characterized both ubiquitous factors that 0bind to diverse regulatory elements and are present in nuclear extracts from many different cell types and unique factors that bind to few promoters or enhancers and are
  • TFs Transcription factors
  • oncogenes such as the fos protein (Lucibello et al., 1988, Oncogene 3_ : 3 ⁇ 52 ) and 0 v-jun protein (Bos et al., 1988, Cell 52_:705-712) .
  • a TF has been found to be associated with the epidermal growth factor (EGF) receptor gene (Kageyama et al., 1988, Proc. Natl. Acad. Sci. U.S.A. 8_5:5016-5020) .
  • Other TFs have been observed to function in a tissue specific manner, such as 5 lymphoid-specific TFs (Muller et al., 1988, Nature
  • c-fos RNA was induced in rat brain by seizures. 5
  • viral TF or cellular TFs which interact with viral promoter/enhancer elements have been identified. These include TF derived from SV40, which appears to activate the SV40 late promoter in vitro (Beard and Bruggmann, 1988, J. Virol. 6_2_:4296-4302) , and nuclear 0 factor EF-C, which occurs in human HepG2 liver cells and in other nonliver cell lines and which is observed to bind to the hepatitus B virus and polyoma virus transcriptional enhancer regions in vitro (Ostapchuk et al., 1989, Mol. Cell. Biol.
  • Another interesting TF is the 5 papillomavirus E2 transactivator protein.
  • the E2 open reading frame of bovine papillomavirus type 1 encodes at least three TFs. These include positive as well as negative regulators of transcription (Lambert et al., 1987, cell 5_0: 64-78; McBride et al., 1988 EMBO J. 7:533-540; 5 Lambert et al., 1989, J. virol 63:3151-3154) .
  • HIV-1 Human immunodeficiency virus type 1
  • AIDS acquired immunodeficiency syndrome
  • LTR long terminal repeat
  • SP1, TATA negative regulatory enhancer
  • TAR regions Five regions of the HIV-1 long terminal repeat (LTR) region, including the negative regulatory enhancer, SP1, TATA, and TAR regions, have been shown to be important in the transcriptional regulation of HIV genes (Garcia et al., 1989, EMBO J. 8:765-778; Harrich et al., 151989, J. Virol. 6_3_:2585-2591) .
  • LTR long terminal repeat
  • the enhancer element has been found to contain two copies of the sequence GGGACTTCC (which shares homology with the eukaryotic TF NF-kappa B) ; the TAR region, located between -17 and +44 in the viral genome, contains two copies of the sequence CTCTCTGG ( u et 0 al., 1988, EMBO J. 7:2117-2130) .
  • Cellular protein EBP-1 has been observed to bind to the enhancer region, whereas cellular protein UBP-1 appears to bind to the TAR region (Wu et al., ibid) .
  • a protein encoded by the human T cell leukemia virus I (HTLV-I) tax gene has been found to 5 interact with the HIV-1 enhancer region.
  • HIV-1 transcriptional transactivator protein tat has also been shown to interact with the TAR region (Arya et al., 1985, Science 229: 69-73; Fisher et al., 1986, Nature 320:367- 371; Kao et al., 1987, Nature 330:489-493) .
  • Three SP-1 binding sites in the HIV LTR were found to be important to iri vitro transcription from the HIV-LTR promoter. Harrick et al. (1989, J. Virol. 6_3:2585-2591) observed that mutagenesis of the HIV-1 LTR
  • SP-1 binding sites resulted in an increase of tat-induced gene expression.
  • SP-1 is a ubiquitous factor which increases transcription of RNA pclymerase II 10 to 50 fold from promoters that contain one or more hexanucleotide sequences, GGGCGG, called GC boxes (Gidoni, supra and
  • the EIB transcriptional unit promoter has only a single SPl binding site and a TATA box (Wu et al., 1987, Nature, 326:512-515). Deletion and linker scan substitution of the SPl site in the adenovirus EIB promoter produced mutant viruses that yielded only 13 to 20% of the basal transcription of wild type virus after infection of
  • HeLa cells (Wu, supra) .
  • Antisense oligonucleotides bind to complementary mRNA sequences and block translation or cause cleavage of the double stranded duplexes formed (Stein, C. A. and Cohen, J. S., 1988, Cancer Res., 48:2659-2668).
  • This approach can decrease expression of specific genes but cannot be generally applied because of limited binding of the oligonucleotide to mRNA regions of strong secondary structure, poor transport into cells and rapid degradation.
  • More stable chemically modified oligonucleotide have multiple stereoisomers that may decrease binding to mRNA (Zon, 1988, supra) .
  • the present invention relates to novel methods for contro]ling gene expression in which double stranded oligonucleotides are used to compete for the binding of nuclear factors to specific cellular transcriptions] control elements.
  • the invention is based in part on the 5 discovery that oligonucleotides containing a GC box can specifically inhibit transcription of EIB.
  • an oligonucleotide comprising one or, preferably, more than one binding site for a transcription factor may be used to 0 inhibit the transcription of genes under the control of promoter/enhancer elements which bind to said transcription factor.
  • the transcription factor is a viral transcription factor, and the method of the invention may be used in the treatment of 5 viral diseases, such as retroviral diseases, in humans or animals.
  • the transcription factor binds to a control element of an oncogene or growth factor, and the method of the invention may be used in the treatment or prevention of cancer.
  • the nucleosides of the oligonucleotide arR joined by phosphorothioate linkage.
  • a oligonucleotide comprising one or more than one binding site for a transcription factor may be used to increase the transcription of genes normally repressed by said transcription factor.
  • oligonucleotides containing a SPl binding sequence may be used to inhibit transcription of EIB.
  • oligonucleotides may comprises multiple copies of SPl binding sequences.
  • FIG. 1 Nucleotide sequence of the EIB transcriptional unit and the inhibitor oligonucleotides.
  • the first line is the promoter region of EIB with the GC and TATA boxes underlined.
  • the sequences of the annealed 14 mers and 28 mers are shown on the next 4 lines oriented below the homologous GC box in the EIB promoter.
  • the transcribed portion of EIB (+1 to +52) is shown on the next line with the synthetic oligonucleotide used for the primer extension oriented below.
  • FIG. 1 Radioautograph of a transcription assay.
  • the heavy bands at the bottom are the 20 base oligomer used for the primer extension.
  • the 50 base bands are the expected transcript for the EIB unit shown in Fig. 1.
  • Reactions in lane 1 and 2 contained no competing oligonucleotides.
  • Lanes 3 and 4 contained 0.22 and 0.87 ⁇ g 14 mer, SP1S.
  • Lanes 5 and 6 contained 0.2 and 1.0 ⁇ g of the 28 mer, 17/19; lanes 7, 8 and 9 contained 0.12, 0.8 and 1.0 ⁇ g of 21/25; and lanes 10, 11 and 12 contained 0.1, 0.8 and 1.0 ⁇ g of 18/20.
  • M is a standard lane of molecular weight markers containing labeled Hpall digest of pBR322.
  • Figure 3. Inhibition of transcription by varying concentrations of oligonucleotides with one, two or three SPl sites. The lines are best fit regressions through the data.
  • Figure 4 Uptake of radiolabelled phosphodiester-1inked (open circles) and phosphorothioate linked (closed circles) oligonucleotides by MOLT 4 cells.
  • the present invention relates to novel methods for controlling gene expression i-n which double stranded oligonucleotides are used to competitively inhibit the .binding of transcription factors to specific transcriptional control elements in DNA.
  • transcription factors which may be inhibited according to the invention
  • identification of oligonucleotides that may be used to inhibit transcriptional factor binding to control elements oligonucleotides of the invention
  • utility of the invention oligonucleotides of the invention
  • double stranded DNA oligonucleotides may be used to inhibit any transcriptional factor which influences transcription by binding to controlling elements of a gene.
  • the transcriptional factor acts to increase transcription of a gene, for example, by binding to a promoter/enhancer element.
  • the transcriptional factor acts to repress transcription of a gene.
  • transcriptional repressers include, but are not limited to, the bovine papillomavirus E2 gene.
  • transcription factors which increase transcription include, but are not limited to, those listed in Table I.
  • oligonucleotides may be used to inhibit the function of transcription factors which are capable of increasing transcription by competing with native DNA binding sites for transcription factor, thereby effectively inhibiting the transcription of a gene (or genes) which is (are) influenced by said transcription factor.
  • double stranded DNA oligonucleotides which bind to a transcription factor which induces the transcription of a viral gene may compete with viral promoter/enhancer elements for transcription factor binding and thereby effectively inhibit the transcription of viral gene sequences.
  • double stranded DNA oligonucleotides may be used to inhibit the function of transcription factors which are capable of repressing transcription by competing with native DNA binding sites for the repressor, thereby effectively increasing the transcription of a gene (or genes) which is (are) influenced by said transcription factor.
  • double stranded DNA oligonucleotides which bind to a viral repressor protein which normally renders a viral gene or genes functionally inactive may be used to activate the expression of these viral genes in a controlled manner; this method may prove useful in the study of latent viral infection in animal models.
  • a particular transcription factor need not be characterized in order to be inhibited according to the present invention. It would be sufficient for a DNA sequence which influences transcription to be identified.
  • the transcription of a particular gene under study may be found to be controlled by a mechanism which includes the presence of a particular DNA sequence; such a sequence might be identified by studying mutations of the gene and its surrounding DNA sequences. Mutation of a sequence important in promoting transcription (a promoter element) may be found to result in a relative decrease in the transcription of a particular gene (either the gene naturally associated with the promoter element or 5 a reporter gene put under the control of the promoter element) .
  • DNA sequences which bind to potential transcription factors may be identified by footprinting techniques or gel retardation analysis using standard techniques known in the art. The DNA sequence of the
  • binding site may then be determined using standard sequencing techniques (for example, Sanger et al., 1979, Proc. Natl. Acad. Sci. U.S.A. 72:3918-3921) .
  • DNA sequences which bind to repressor transcription factors may be identified in an analogous
  • Oligonucleotides which may be used to inhibit transcription factor binding to control elements may be identified by determining whether said oligonucleotides (a) bind to said transcription factor and/or (b) inhibit the function of said transcription factor.
  • - oligonucleotide may be tested directly for binding to the TF.
  • TF may be immobilized, and then exposed to labeled oligonucleotide, upon which selective retention of labeled oligonucleotide to TF could be measured.
  • oligonucleotide may be included in the reaction in which TF (in purified or unpurified form) is allowed to bind to DNA; the oligonucleotide may be observed to competitively inhibit the binding of TF to its target sequence, thereby diminishing the appearance of a clear "footprint.”
  • characterized or uncharacterized, purified or unpurified TF may be tested for the ability to bind an oligonucleotide using gel retardation analysis (Barberis et al., 1987, cell 5JD:347-359) .
  • an oligonucleotide could be exposed to purified TF or, alternatively, TF as found in a mixture (e.g. a nuclear extract) under conditions which may allow binding of TF to the oligonucleotide.
  • a mixture e.g. a nuclear extract
  • oligonucleotides may be tested for the ability to inhibit the function (e.g. as inducer or repressor) of said transcription factor.
  • Oligonucleotides may be evaluated using transcription systems in vitro or in vivo which comprise a control element which is believed to interact with a transcription factor and which controls the expression of a test gene.
  • Nuclear extracts may be prepared from actively growing cells as described in Section 6.1.3., infra. Transcription mixtures may contain the following: about 0.5-1.2 ⁇ g of DNA comprising a test gene under the control of an element which is believed to bind to a transcription factor in a final concentration of 226 M HEPES pH 7.9, 48 M KC1, 6 mM MgCl ?
  • oligonucleotide 0.1 to 1.0 ⁇ g/reaction; preferably, a range of amounts are tested.
  • Reaction may be started by addition of extract (generally about 1-2 ⁇ g protein/ ⁇ l nuclear extract) incubated 30-90 minutes at 30°C, and terminated by addition of a mixture of 175 ⁇ g of 200 mM NaCl,20 mM EDTA and 1% sodium dodecyl sulfate, 20 g purified yeast tRNA, followed by extraction with 100 ⁇ g phenol and 100 ⁇ g chloroform-isoamyl alcohol (19:1) to each tube.
  • extract generally about 1-2 ⁇ g protein/ ⁇ l nuclear extract
  • the oligonucleotides in the aqueous phase may be precipitated with 0.5 M NH acetate and 3 volumes of ethanol, resuspended in 200 ⁇ g 0.3 M Na acetate pH6, reprecipitated with 3 volumes of ethanol and dried in a vacuum centrifuge.
  • the mRNA products of transcription in this, or any transcription assay may be analyzed by any method known in the art, including, but not limited to, Northern blot analysis and/or quantitative hybridization (e.g. hybridization of labeled mRNA to DNA immobilized on filters) .
  • a primer extension assay such as that developed with EIB for analysis of transcriptional factors (see Section 6.1.2. and 6.2.1., infra) may be used; in particular, the residue from the aqueous phase of the transcription reaction may be dissolved in 10 ⁇ g of 0.25 M KC1 in TE buffer containing
  • the solution may then be cooled and the primer extended by incubation for about 30 minutes at 37°C in a solution containing 14 mM tris buffer pH8, 7 mM MgCl , 3.5 mM DTT, 0.2 mM each of dATP, dGTP, dCTP and DTTP, 7 ⁇ g/ml of actinomycin D and 0.085 U/ ⁇ l Maloney urine leukemia virus reverse transcriptase in a final volume of about 35 ⁇ l.
  • the nucleic acid may be precipitated with 0.3 M Na acetate and 3 volumes of ethanol, dried, and dissolved in 10 ⁇ g buffered formamide containing bro phenol blue and xylene cyanol, then subjected to polyacrylamide gel electrophoresis at 300-400V in a 8 M urea, 10% acrylamide, 0.3% bis-acrylamide gel. Bands may be cut from dried gels -and quantified by liquid scintillation counting.
  • the activity of a TF may be evaluated in vitro in a system which comprises the TF and a transcription template that includes a reporter gene under the control of a promoter which binds to said TF.
  • the effect of test oligonucleotides on reporter gene expression may be expected to reflect the effectiveness of the oligonucleotide in inhibiting TF binding.
  • an .in vitro system may be utilized in which HIV tat protein is the TF and the transcription template is a reporter gene, such as the gene encoding chloramphenicol acetyltransferase (CAT) , under the control of the HIV-1 LTR promoter.
  • CAT chloramphenicol acetyltransferase
  • double stranded oligonucleotides comprising one or more SPl or NF kappa B sites, or both, may inhibit tat activity.
  • transcription may be carried out in isolated nuclei or whole cells which comprise the control element which is believed to bind to transcription factor, such that inhibition of TF binding to the control element and consequent inhibition of TF function is detectable.
  • the amount of RNA transcribed from a gene controlled by said control element may be measured in the presence or absence of oligonucleotide in pulse-labeling experiments of cells or isolated nuclei.
  • a protein product of a gene controlled by said control element may be monitored in the presence or absence of oligonucleotide; proteins suitable may include, but are not limited to, standard reporter genes such as chloramphenicol/acetyltransferase, / 9-galactosidase, luciferase, etc.
  • a transcription template comprising a reporter gene and a control element may be transfected into cells which contain a transcription factor of interest.
  • the effect on reporter gene expression of exposing such transfected cells to oligonucleotides of the invention may be used .as a measure of the effectiveness of the oligonucleotides in entering the cells and inhibiting TF binding.
  • Any suitable reporter gene may be used.
  • reporter gene refers to any detectable gene product; reporter gene products which are easily and inexpensively detected are preferred. It may be desirable, under certain circumstances, to use a reporter gene that encodes a product for which a highly sensitive assay is available.
  • a highly sensitive radioi munoassay is available for human growth hormone (HGH) .
  • HGH human growth hormone
  • a specific assay system such as HeLa cells which express tat protein transfected with a transcription template that comprises the gene for HGH under the control of the HIV-1 LTR, may be used to test the efficacy of oligonucleotides inhibiting tat binding in a non-limiting embodiment of the present invention.
  • Oligonucleotide sequences which may represent TF binding sites may be identified by techniques including mutational analysis, footprinting studies, and gel retardation analysis as described above. Oligonucleotide sequences which have been associated with TF binding and HIV-1 transcriptional regulation, and which may be used according to the invention, are presented in Table II. Binding site sequences which are as yet to be characterized are also provided for by the invention.
  • Oligonucleotides may be synthesized using any technique used in the art.
  • the present invention construes oligonucleotides to mean a series of nucleotides linked together, and includes nucleotides linked in a standard 5'-3' phosphodiester linkage and also molecules comprising a methylated nucleotide or nucleotide similarly modified, or any nucleoside analogue or enantiomer, or nucleosides joined by phosphorothioate linkage or molecules which comprise a variety of chemical linkages (e.g. phosphodiester and phosphorothioate) .
  • an oligonucleotide comprises at least one phosphorothioate linkage.
  • Phosphorothioate modification of an oligonucleotide has been observed to enhance TF inhibition, and represents a preferred 5 embodiment of the invention. Furthermore, as shown in example section 7, infra, phosphorothioate-linked oligonucleotides are able to reach higher concentrations in cells compared to phosphodiester-linked oligonucleotides. In a preferred embodiment, phosphorothioate- 0 linkages may be enzymatically introduced into double- stranded oligonucleotides of the invention.
  • phosphorothioate linked oligonucleotides may be prepared by a reaction comprising commercially available alpha-thio-nucleotides and primerr, 5 which include putative TF-binding sequences, utilizing polymerase chain reaction (PCR) technology (Saiki et al., 1985, Science 230:1350-1354) .
  • PCR polymerase chain reaction
  • the oligonucleotides of the invention comprise at least a portion which is double stranded, and include 0 oligonucleotides with blunt (double-stranded) ends as well as oligonucleotides with single stranded "overhangs", in which the ends of the molecule are extensions of single- stranded nucleotide sequence beyond a double stranded nucleotide sequence beyond a double stranded region. It 5 has been observed that oligonucleotides which comprise a single-stranded "overhang" are more effective in inhibiting TF binding, and accordingly represent preferred embodiments of the invention.
  • oligonucleotides of the invention comprise ⁇ one or, preferably, more than one binding site for a transcription factor. Oligonucleotides of the invention may also comprise binding sites for more than one specie;;
  • transcription factor 5 of transcription factor; this may be particularly useful if two transcription factors function coordinately to control Transcription of a gene.
  • oligonucleotides of the invention may be linked to molecules which may aid the ability of the oligonucleotides to physically reach the transcriptional apparatus. Examples may include molecules with hydrophobic regions which may facilitate the penetration of the oligonucleotide through the cell membrane.
  • the oligonucleotides may be linked to a nuclear localization signal (such as is utilized by SV40) .
  • oligonucleotides of the invention may be targeted toward a particular cell type or tissue (e.g. virus-infected cells) by an antibody specific for that cell type or tissue; oligonucleotide may be released from the antibody and then function to inhibit transcription in a particular cell type or tissue.
  • oligonucleotides may be comprised in liposomes or microcapsules, which also offers the advantage of preventing degradation of the oligonucleotides prior to cellular uptake.
  • the present invention may be used to either increase or decrease the transcription of a gene or genes, the transcription of which is regulated by the interaction of a control element of the gene or genes and a transcription factor.
  • oligonucleotides are used to inhibit the binding of the transcription factor to its control element. If the transcription factor normally acts to increase transcription, then transcription may effectively be decreased by competing oligonucleotides. If the transcription factor normally acts to repress transcription, then transcription may be effectively increased by competing oligonucleotides.
  • the transcription factor to be inhibited is SPl.
  • the transcription factor is the HIV-1 tat protein, or, alternatively, the HTLV-I tax protein, or NF-kappa B.
  • Transcription factors may specifically influence the expression of some genes, but not others; for example, some TFs specifically influence the transcription of viral genes, and others specifically influence the transcription* of genes in certain tissues (including, but not limited to, pituitary or lymphoid cells) .
  • the specificity of TFs enables the manipulation of the transcription of genes of interest via the specific inhibition of TF binding by oligonucleotides.
  • oligonucleotides For TFs which are not optimally specific, it may be desirable to direct oligonucleotides to a subpopulation of cells which utilize the TF of interest.
  • a cellular TF may induce the transcription of viral as well as cellular genes; if oligonucleotide were supplied to all cells in the body of an organism, transcription of viral and cellular genes would be inhibited in a manner potentially harmful to the organism as a whole. If, however, oligonucleotides were delivered only to virus infected cells (e.g. via antibody to a viral antigen present on the surface of infected cells, or an antibody- targeted liposome or microcapsule) , then only infected cells would be affected.
  • the use of oligonucleotides which comprise double-stranded regions offers advantages over the use of other competitive oligonucleotides such as antisense RNA.
  • Such advantages include increased stability, particularly in the case of oligonucleotides comprising a phosphorothioate linkage and the fact that lower concentrations of oligonucleotide may be required to effectively compete with transcription factors (present in low concentration) as compared to the concentration required to compete with mRNA (present in higher concentrations) , (See Section 6.3., infra).
  • the present invention may be utilized to control the transcription rates of any gene influenced by transcription factor binding, including cellular genes and viral genes.
  • Clinical applications may include, but are not limited to, decreasing the imbalance of expression of globin genes in thalassemia, inhibiting the transcription of oncogenes in the treatment or prevention of malignancy inhibiting the production of growth hormone in acromegaly and inhibiting the transcription of oncogenes in the treatment or prevention of malignancy.
  • the present invention also provides for treatment of a wide variety of viral diseases, including, but not limited to, AIDS, HTLV-I infection, and papillomavirus infection.
  • the EIB transcriptional unit was the Xbal-Sacl fragment, nucleotides 1336 to 1767 of the adenovirus-2 genome inserted into a PUC18 vector (Wu, supra and Gineras et al., 1982, J. Biol. Chem. 257:13475-13491). A single stranded oligonucleotide complementary to nucleotides 1730 to 1750 was used for primer extension analysis of EIB transcription.
  • the vector and primer were gifts from M. Schmidt and A. Berk, Molecular Biology Institute, University of California, Los Angeles.
  • the pDUG plasmid consists of the E fragment of a Ball digest of the adenovirus-2 genome inserted into a PHC 314 vector.
  • the E fragment contains the major late promoter of the adenovirus but no SPl binding site (Leong et al., 1988, Mol. Cell. Biol. 8:1785-1774. 5
  • OLIGONUCLEOTIDE SYNTHESIS Complementary 14 mers identical to the ones used by Kadonaga, J. T. and Tjian, R. , 1986, Proc. Natl. Acad. Sci. (USA), 83:5889-5893 for purification of the SPl 10 transcriptional factor were chemically synthesized by D. Glick, Department of Biochemistry, University of California, Los Angeles. Additional oligonucleotides were .synthesized by J. Tomich, Division of Genetics, Children's Hospital of Los Angeles, or the Microchemical Core Facility
  • Complementary 28 mers containing the SPl binding site were synthesized.
  • oligonucleotides were synthesized corresponding to binding sites for AP2, a TATA box and a low affinity binding sequence for SPl from SV40 0 (Kadonaga, J. T. and Tjian, R. , 1986, Proc. Natl. Acad.
  • oligonucleotide containing both an SPl site and a TATA box separated by the same number of nucleotides as in the EIB transcriptional unit was synthesized to look for a possible interaction between 5 the two factors.
  • Phosphorothioate analogues of the blunt ended oligonucleotide set containing 2 SPl sites, SP1X2B, and a self-complementary 26 mer containing the Pvul restriction site were synthesized by hydrogen phosphonate chemistry on an Applied Biosystems DNA synthesizer and by single step sulfurization following chain assembly. All oligonucleotides were purified and annealed as described (Harrington et al., 1988, Proc. Natl. Acad. Sci. (USA), 85:2066-2070) .
  • the sequence of the EIB transcriptional unit, 14 mers, 28 mers and primer are shown in Fig. 1.
  • the SPl binding site and TATA, box are underlined. Transcription begins at residue +1 (Wu, supra) .
  • the sequences of other oligonucleotides are listed in Table III.
  • SP1X2B GATCGGGGCGGGGCGGGGGGCGGGGC CTAGCCCCGCCCCGCCCCCCGCCCCG
  • EIB plasmid DNA or pDUG plasmid DNA in a final concentration of 26 mM HEPES pH 7.9, 48 mM KC1, 6 mM MgCl 2 , 9.6% glycerol, 0.1 M EDTA, 0.6 mM each of ATP, GTP CTP and UTP, 0.5 mM dithiothreitol (DTT) and 0.5 mM phenylmethylsulfonylfluoride along with varying amounts of
  • MMLV 35 transcriptase (Bethesda Research Laboratories, Gaithersburg, MD, USA) in a final volume of 35 ⁇ l.
  • the nucleic acid was precipitated with 0.3 M Na acetate and 3 volumes of ethanol, dried, and dissolved in 10 ⁇ l buffered formamide containing bromphenol blue and xylene cyanol. They were electrophoresed at 300-400 V in a 8 M urea, 10% acrylamide, 0.3% bis-acryla ide gel. Transcription of the EIB template resulted in a 50 nucleotide band.
  • the bands were cut from the dried gels and quantified by liquid scintillation counting.
  • the degree of polymerization was studied by incubation of [ 32P]-labelled oligonucleotides under the same conditions as for in vitro transcription. After precipitation with ethanol, the dried residue was dissolved in running dye and electrophorsed under non-denaturing' conditions at 300-400 V in a 10% acrylamide, 0.3% bis- acrylamide gel. All bands were cut from the dried gels and quantified by liquid scintillation counting.
  • transcript 50 nucleotide band.
  • the amount of transcript increased with extract concentration, time to 90 min. and template concentration to 21 ⁇ l/ml (11 nM) .
  • template concentration 21 ⁇ l/ml (11 nM) .
  • transcription was lower at a template concentration of 40 ⁇ g/ml.
  • Two ⁇ g/ml ⁇ -amanitin (Sigma, St. Louis, MO) inhibited the transcription indicating that it was dependent on RNA polymerase II.
  • MgCl_ concentrations were optimal between 6 and 7.5 mM with different extracts.
  • Sper idine 1-4 mM inhibited transcription and decreased the effect of ⁇ -amanitin.
  • the amount of competitor oligonucleotide required to inhibit to 20% and 50% of control was determined from regression lines using Sigmaplot statistical software, The amount required for 50% inhibition is also shown as oligonucleotide concentration and the molar ratio of oligonucleotide to template DNA.
  • Table IV shows the concentrations of the various oligonucleotides needed to decrease EIB transcription to 20% and 50% of the control without competitor oligonucleotides.
  • An IC20 value gives an approximate concentration needed to achieve maximal inhibition.
  • the IC50 is also expressed as the molar ratio of oligonucleotide to template DNA.
  • the IC50 was 0.87 ⁇ M for SP1S and SP1B, 14 mers with sticky and blunt ends.
  • An oligonucleotide with a low affinity SPl binding site, SP1L required an IC50 of 2.80 ⁇ M.
  • the addition of a single methylated cytosine in 21/25 did not affect transcription of EIB, while the completely methylated 28 mer, 18/20, had an IC50 of 0.74 ⁇ M.
  • the effect of a single methylation was minimal on the ability of the synthetic oligonucleotides to compete in this transcription assay.
  • a completely methylated oligonucleotide had a decreased ability to compete for SPl.
  • oligonucleotides with two and three SPl sites separated by 12 nucleotides were synthesized (Fig. 3). IC50 concentrations for the sticky ended oligonucleotides with one, two or three SPl sites were 0.87, 0.35 and 0.07 ⁇ M, respectively. IC50 concentration for the blunt ended oligonucleotide with two SPl sites was 0.16 ⁇ M. Inhibition of transcription to 20% of control followed the same pattern.
  • oligonucleotides competed for binding factors much less efficiently. IC50 concentrations ranged from 1.97 ⁇ M to 2.05 ⁇ M, approximately threefold greater than the amount required for the oligonucleotides with high. affinity SPl sites. The competition by the AP2 oligonucleotide is consistent with the interactions reported between SPl and AP2 with DNAase protection assays (Mitchell et al. , 1987, Cell, 50:847-861).
  • SP1TATA Another oligonucleotide, SP1TATA, contained a single SPl binding site and a TATA binding site with the same spacing as in the EIB promoter. This oligonucleotide was only slightly better inhibitor than an oligonucleotide with only an SPl site.
  • Oligonucleotides with blunt ends or with four base complimentary overhangs showed differences in the shape of the inhibition curves, but similar IC50 and IC20 values. During incubation with nuclear extracts, the sticky ended oligonucleotides might form higher molecular weight structures more readily than the blunt ended ones.
  • the double stranded phosphorothioate was a more effective inhibitor than SP1X2B.
  • a control 26 mer phosphorothioate containing a Pvul site did not inhibit EIB transcription at concentrations of l ⁇ g/25 ⁇ l.
  • Oligonucleotides with sticky ends were generally 0 more effective in inhibiting transcription than blunt ends. This was not due to polymerization but may be due to better ⁇ resistance to degradation.
  • 35 steroisomers do not appear to prevent SPl binding.
  • the improved effect may be due to resistance to nucleases present in cell extracts.
  • Double stranded oligonucleotides are designed to bind nuclear binding factors rather than mRNA molecules. In most cases, there are few copies of transcriptional elements for a specific gene and few molecules of nuclear molecules of nuclear binding factors relative to the number of RNA transcripts. This advantage is illustrated by our data showing that the concentrations of double stranded oligonucleotides that inhibit transcription are 10 to 100 times lower than the reported concentrations of antisense oligonucleotides needed to block expression of various genes _in vitro (Stein, 1988, supra) . Our experiments have defined some specific parameters for double stranded oligonucleotides to achieve optimal inhibition of .in vitro transcription of EIB. Similar experiments with more complex promoters may define interactions of their nuclear binding factors.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Zoology (AREA)
  • Biotechnology (AREA)
  • Wood Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • Biochemistry (AREA)
  • Virology (AREA)
  • General Health & Medical Sciences (AREA)
  • Microbiology (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Plant Pathology (AREA)
  • AIDS & HIV (AREA)
  • Immunology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Analytical Chemistry (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

Nouveaux procédés de commande de l'expression de gènes, consistant à utiliser des oligonucléotides bicaténaires afin d'inhiber l'interaction de facteurs de transcription à l'aide d'éléments de commande de transcription dans de l'ADN. Les procédés de l'invention sont particulièrement utiles pour inhiber sélectivement la transcription de gènes et d'oncogènes viraux, et on peut les utiliser dans le traitement d'une variété de maladies virales. Selon les modes de réalisation préférés de l'invention, les nucléosides sont reliés par une liaison phosphorothioate.
PCT/US1991/000635 1990-01-30 1991-01-30 Inhibition de transcription a l'aide d'oligonucleotides bicatenaires WO1991011535A1 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
US47213790A 1990-01-30 1990-01-30
US472,137 1990-01-30
USNOTFURNISHED 2006-10-30

Publications (1)

Publication Number Publication Date
WO1991011535A1 true WO1991011535A1 (fr) 1991-08-08

Family

ID=23874341

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1991/000635 WO1991011535A1 (fr) 1990-01-30 1991-01-30 Inhibition de transcription a l'aide d'oligonucleotides bicatenaires

Country Status (1)

Country Link
WO (1) WO1991011535A1 (fr)

Cited By (20)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0523140A4 (en) * 1990-03-21 1993-06-16 Isis Pharmaceuticals, Inc. Reagents and methods for modulating gene expression through rna mimicry
WO1995001429A1 (fr) * 1993-06-30 1995-01-12 Rhone-Poulenc Rorer S.A. Compositions pharmaceutiques et leur utilisation, notamment pour le traitement des maladies neurodegeneratives
WO1995012415A1 (fr) * 1993-11-05 1995-05-11 Isis Pharmaceuticals, Inc. Modulation de l'expression de molecules d'adherence intercellulaire vasculaire par interactions de nouveaux oligonucleotides
US5532130A (en) * 1993-07-20 1996-07-02 Dyad Pharmaceutical Corporation Methods and compositions for sequence-specific hybridization of RNA by 2'-5' oligonucleotides
US5747253A (en) * 1991-08-23 1998-05-05 Isis Pharmaceuticals, Inc. Combinatorial oligomer immunoabsorbant screening assay for transcription factors and other biomolecule binding
WO1998045468A1 (fr) * 1997-04-08 1998-10-15 The United States Of America As Represented By The Administrator Of The National Aeronautics And Space Administration (Nasa) Elaboration de proteines fonctionnelles : equilibre entre contrainte tangentielle et gravite
US5955644A (en) * 1996-08-08 1999-09-21 M.D. Anderson Cancer Center Ku deficient cells and non-human transgenic animals
US5990090A (en) * 1993-09-20 1999-11-23 The University Of Michigan Methods and compositions for treatment of diseases
EP0733370A4 (fr) * 1993-10-29 1999-12-29 Shunichi Shiozawa Inhibiteur antagoniste de la croissance des cellules mesenchymateuses
US6057489A (en) * 1996-09-12 2000-05-02 M.D. Anderson Cancer Center MmRad51-deficient cells and transgenic mice
EP0732929A4 (fr) * 1993-10-29 2001-11-14 Victor J Dzau Utilisation therapeutique de leurres d'elements cis in vivo
US6776986B1 (en) 1996-06-06 2004-08-17 Novartis Ag Inhibition of HIV-1 replication by antisense RNA expression
US6911541B2 (en) 2000-08-30 2005-06-28 North Carolina State University Promoter fragment that is recognized by the product of the tobacco Nic gene
US7189570B2 (en) 2000-11-07 2007-03-13 North Carolina State University Putrescine-n-methyltransferase promoter
WO2007038658A2 (fr) 2005-09-26 2007-04-05 Medarex, Inc. Conjugues anticorps-medicament et leurs methodes d'utilisation
US7304220B2 (en) 1997-06-12 2007-12-04 North Carolina State University Regulation of quinolate phosphoribosyl transferase expression
US7507420B2 (en) 2001-05-31 2009-03-24 Medarex, Inc. Peptidyl prodrugs and linkers and stabilizers useful therefor
US7517903B2 (en) 2004-05-19 2009-04-14 Medarex, Inc. Cytotoxic compounds and conjugates
US7615538B2 (en) 1993-10-29 2009-11-10 Shunichi Shiozawa Method for therapy of rheumatoid arthritis
EP3255144A1 (fr) 2007-08-10 2017-12-13 E. R. Squibb & Sons, L.L.C. Construction de type recombineering pour la préparation de souris transgeniques capables de produire des immunoglobulines humaines.

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
CELL, Vol. 50, issued 03 July 1987, LAMBERT et al., "A Transcriptional Repressor Encoded by BPV-1 Shares a Common Carboxy Terminal Domain with the E2 Transactivator", pages 69-78. *
NATURE, Vol. 312, issued 29 November 1984, GIDOI et al., "Multiple Specific Contacts Between a Mammalian Transcription Factor and its Cognate Promoters", pages 409-413. *
NUCLEIC ACIDS RESEARCH, Vol. 17, No. 21, issued 1989, LATCHMAN et al., "The Different Competitive Abilities of Viral TAAGARAT Elements and Cellular Octamer Motifs, Mediate the Induction of Viral Immediate-Early Genes and the Repression of the Histone H2B Gene in Herps Simplex Virus Infected Cells", pages 8533-8542. *
SCIENCE, Vol. 229, issued 05 July 1985, ARYA et al., "Trans-Activator Gene of Human T-Lymphotropic Virus Type III (HTLV-III)", pages 69-73. *
THE EMBO JOURNAL, Vol. 7, No. 7, issued 1988, WU et al., "Purification of the Human Immunodeficiency Virus Type 1 Enhancer and TAR Binding Proteins EBP-1 and UBP-1", pages 2117-2129. *
THE EMBO JOURNAL, Vol. 8, No. 3, issued 1989, GARCIA et al., "Human Immunodeficiency Virus Type 1 LTR TATA and TAR Region Sequences Required for Transcriptional Regulation", pages 765-778. *

Cited By (36)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0523140A4 (en) * 1990-03-21 1993-06-16 Isis Pharmaceuticals, Inc. Reagents and methods for modulating gene expression through rna mimicry
US5736294A (en) * 1990-03-21 1998-04-07 Isis Pharmaceuticals, Inc. Reagents and methods for modulating gene expression through RNA mimicry
US5747253A (en) * 1991-08-23 1998-05-05 Isis Pharmaceuticals, Inc. Combinatorial oligomer immunoabsorbant screening assay for transcription factors and other biomolecule binding
WO1995001429A1 (fr) * 1993-06-30 1995-01-12 Rhone-Poulenc Rorer S.A. Compositions pharmaceutiques et leur utilisation, notamment pour le traitement des maladies neurodegeneratives
FR2707880A1 (fr) * 1993-06-30 1995-01-27 Rhone Poulenc Rorer Sa Compositions pharmaceutiques et leur utilisation, notamment pour le traitement des maladies neurodégénératives.
EP1454984A1 (fr) * 1993-06-30 2004-09-08 Aventis Pharma S.A. Compositions pharmaceutiques et leur utilisation, notamment pour le traitement des maladies neurodégénératives
AU696644B2 (en) * 1993-06-30 1998-09-17 Aventis Pharma S.A. Pharmaceutical compositions and their use, in particular for the treatment of neurodegenerative diseases
KR100517879B1 (ko) * 1993-06-30 2005-12-16 아방티 파르마 소시에테 아노님 신경 퇴행성 질환의 치료를 위한 제약학적 조성물 및 이들의 사용방법
US5532130A (en) * 1993-07-20 1996-07-02 Dyad Pharmaceutical Corporation Methods and compositions for sequence-specific hybridization of RNA by 2'-5' oligonucleotides
US5990090A (en) * 1993-09-20 1999-11-23 The University Of Michigan Methods and compositions for treatment of diseases
US6821956B2 (en) 1993-10-29 2004-11-23 The Brigham And Women's Hospital, Inc. Therapeutic use of cis-element decoys in vivo
EP0733370A4 (fr) * 1993-10-29 1999-12-29 Shunichi Shiozawa Inhibiteur antagoniste de la croissance des cellules mesenchymateuses
US7615538B2 (en) 1993-10-29 2009-11-10 Shunichi Shiozawa Method for therapy of rheumatoid arthritis
EP0732929A4 (fr) * 1993-10-29 2001-11-14 Victor J Dzau Utilisation therapeutique de leurres d'elements cis in vivo
EP1350514A3 (fr) * 1993-10-29 2004-07-07 The Brigham And Women's Hospital, Inc. Utilisation thérapeutique de leurres d'élements cis in vivo
EP1340505A3 (fr) * 1993-10-29 2004-07-14 The Brigham And Women's Hospital, Inc. Utilisation thérapeutique de leurres d'éléments-cis in vivo
US6774118B1 (en) 1993-10-29 2004-08-10 The Brigham And Women's Hospital, Inc. Therapeutic use of CIS-element decoys in vivo
US6399376B1 (en) 1993-11-05 2002-06-04 Isis Pharmaceuticals, Inc. Modulation of vascular cell adhesive molecule expression through oligonucleotide interactions
US6713621B1 (en) 1993-11-05 2004-03-30 Isis Pharmaceuticals, Inc. Chimeric oligonucleotides for modulating gene expression
WO1995012415A1 (fr) * 1993-11-05 1995-05-11 Isis Pharmaceuticals, Inc. Modulation de l'expression de molecules d'adherence intercellulaire vasculaire par interactions de nouveaux oligonucleotides
US6776986B1 (en) 1996-06-06 2004-08-17 Novartis Ag Inhibition of HIV-1 replication by antisense RNA expression
US5955644A (en) * 1996-08-08 1999-09-21 M.D. Anderson Cancer Center Ku deficient cells and non-human transgenic animals
US6057489A (en) * 1996-09-12 2000-05-02 M.D. Anderson Cancer Center MmRad51-deficient cells and transgenic mice
WO1998045468A1 (fr) * 1997-04-08 1998-10-15 The United States Of America As Represented By The Administrator Of The National Aeronautics And Space Administration (Nasa) Elaboration de proteines fonctionnelles : equilibre entre contrainte tangentielle et gravite
US7408098B2 (en) 1997-06-12 2008-08-05 North Carolina State University Regulation of quinolate phosphoribosyl transferase expression
US7605308B2 (en) 1997-06-12 2009-10-20 North Carolina State University Regulation of quinolate phosphoribosyl transferase expression
US7425670B2 (en) 1997-06-12 2008-09-16 North Carolina State University Methods and compositions for protein production in tobacco plants with reduced nicotine
US7304220B2 (en) 1997-06-12 2007-12-04 North Carolina State University Regulation of quinolate phosphoribosyl transferase expression
US7192771B2 (en) 2000-08-30 2007-03-20 North Carolina State University Plant promoter sequence
US6911541B2 (en) 2000-08-30 2005-06-28 North Carolina State University Promoter fragment that is recognized by the product of the tobacco Nic gene
US7189570B2 (en) 2000-11-07 2007-03-13 North Carolina State University Putrescine-n-methyltransferase promoter
US7507420B2 (en) 2001-05-31 2009-03-24 Medarex, Inc. Peptidyl prodrugs and linkers and stabilizers useful therefor
EP2266986A1 (fr) 2001-05-31 2010-12-29 Medarex, Inc. Cytotoxines, promedicaments, lieurs et stabilisateurs utiles pour ceux-ci
US7517903B2 (en) 2004-05-19 2009-04-14 Medarex, Inc. Cytotoxic compounds and conjugates
WO2007038658A2 (fr) 2005-09-26 2007-04-05 Medarex, Inc. Conjugues anticorps-medicament et leurs methodes d'utilisation
EP3255144A1 (fr) 2007-08-10 2017-12-13 E. R. Squibb & Sons, L.L.C. Construction de type recombineering pour la préparation de souris transgeniques capables de produire des immunoglobulines humaines.

Similar Documents

Publication Publication Date Title
WO1991011535A1 (fr) Inhibition de transcription a l'aide d'oligonucleotides bicatenaires
Kjems et al. Specific binding of a basic peptide from HIV-1 Rev
US5176996A (en) Method for making synthetic oligonucleotides which bind specifically to target sites on duplex DNA molecules, by forming a colinear triplex, the synthetic oligonucleotides and methods of use
EP0375408B1 (fr) Méthode de préparation d'oligonucléotides synthétiques se liant spécifiquement à des cibles sur des molécules d'ADN bicaténaire en formant un complexe tricaténaire colinéaire, les oligonucléotides synthétiques et méthodes d'utilisation
Marciniak et al. Identification and characterization of a HeLa nuclear protein that specifically binds to the trans-activation-response (TAR) element of human immunodeficiency virus.
Lieber et al. Selection of efficient cleavage sites in target RNAs by using a ribozyme expression library
Vegvar et al. Nucleocytoplasmic transport and processing of small nuclear RNA precursors
US6017756A (en) Method and reagent for inhibiting hepatitis B virus replication
EP0638121B1 (fr) Clivage cible d'arn au moyen de ribonuclease eucaryote p et d'une sequence-guide externe
US5989911A (en) Site-specific synthesis of pseudouridine in RNA
US5821046A (en) RNA oligonucleotides that bind HIV tat protein
Keen et al. Transfer of Tat and release of TAR RNA during the activation of the human immunodeficiency virus type‐1 transcription elongation complex
Canonne-Hergaux et al. Interactions of the transcription factor AP-1 with the long terminal repeat of different human immunodeficiency virus type 1 strains in Jurkat, glial, and neuronal cells
GOILA et al. Inhibition of hepatitis B virus X gene expression by novel DNA enzymes
Yee et al. Identification of novel single-stranded d (TC) n binding proteins in several mammalian species
WO1995004818A1 (fr) Procede et reactif servant a inhiber la replication du virus de l'immunodeficience humaine
US5801158A (en) Enzymatic RNA with activity to RAS
US6518417B1 (en) Antisense nucleic acids targeting HBV
Pikaard Ribosomal gene promoter domains can function as artificial enhancers of RNA polymerase I transcription, supporting a promoter origin for natural enhancers in Xenopus.
Joel et al. A nuclear protein with enhanced binding to methylated Sp1 sites in the AIDS virus promoter
Ferguson et al. Complex functional interactions at the early enhancer of the PQ strain of BK virus
AU2001284570B2 (en) New sequences
Marozzi et al. In vitro selection of HIV-1 TAR variants by the Tat protein
Winandy et al. Nuclear factors that bind two regions important to transcriptional activity of the simian immunodeficiency virus long terminal repeat
AU2001284570A1 (en) New sequences

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AU BB BG BR CA FI HU JP KR LK MC MG MW NO PL RO SD SU

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): AT BE BF BJ CF CG CH CM DE DK ES FR GA GB GR IT LU ML MR NL SE SN TD TG

NENP Non-entry into the national phase

Ref country code: CA