WO1994005268A1 - Nouveaux oligonucleotides formant des helices triples, et leurs procedes d'utilisation - Google Patents

Nouveaux oligonucleotides formant des helices triples, et leurs procedes d'utilisation Download PDF

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Publication number
WO1994005268A1
WO1994005268A1 PCT/US1993/008329 US9308329W WO9405268A1 WO 1994005268 A1 WO1994005268 A1 WO 1994005268A1 US 9308329 W US9308329 W US 9308329W WO 9405268 A1 WO9405268 A1 WO 9405268A1
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Prior art keywords
target
steroid hormone
seq
triplex forming
synthetic
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PCT/US1993/008329
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English (en)
Inventor
Ming-Jer Tsai
Michael E. Hogan
Bert W. O'malley
Nancy Hughes Ing
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Baylor College Of Medicine
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Priority to AU48467/93A priority Critical patent/AU4846793A/en
Publication of WO1994005268A1 publication Critical patent/WO1994005268A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • 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
    • C12N15/1138—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 receptors or cell surface proteins
    • 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
    • C12N2310/15—Nucleic acids forming more than 2 strands, e.g. TFOs

Definitions

  • This invention relates generally to the field of triplex forming oligonucleotides. More specifically, the present invention relates to novel triplex forming oligonucleotides that inhibit steroid hormone induced gene transcription. 2. Description of the Related Art
  • Steroid hormones control the growth, differentiation, and function of reproductive and other tissues by affecting gene expression.
  • the female steroids, progesterone and estrogen also influence the growth of many tumors, including those of breast and uterine origin. Therefore, control of steroid hormone action is of major importance, as witnessed by increasing use of antihormones in reproductive control and cancer treatment.
  • progesterone action involves binding and activation of a nuclear receptor which binds a specific DNA sequence present in the promotors of progesterone-responsive genes to activate their transcription.
  • the most common therapeutic agents aimed at disrupting progesterone action are progesterone analogs that bind, but do not activate, the receptor. When given systemically, these agents affect all progesterone-regulated body processes and cause certain unwanted side effects.
  • Triplex formation is a relatively new concept as a basis for pharmacological therapeutics. Recently, triplex forming oligonucleotide (TFO) reagents which bind to DNA in a site selective manner have been developed. This work has demonstrated that synthetic single stranded DNA oligonucleotides targeted to repeating A or G segments within a
  • DNA gene can form stable triplexes at acidic pH.
  • the formation of these triple helices is based upon hydrogen bonding of T to the AT bases in a DNA double helix or duplex and a protonated cytosine (C+) to a GC duplex.
  • C+ protonated cytosine
  • triplex forming oligonucleotides of this new type may be prepared by scanning the genomic duplex DNA and identifying nucleotide target sequences of greater than about 20 nucleotides having about at least 65% purine bases on one strand; and synthesizing the synthetic oligonucleotide complementary to the identified target sequence, where the synthetic oligonucleotide has a G when the complementary location in the DNA duplex has a GC base pair and has a T when the complementary location of the DNA duplex has an AT base pair.
  • the triplex strategy has certain advantages, e.g., fewer and less regenerative targets (single copy genes vs. multiple copy mRNAs).
  • TFOs When TFOs are targeted to a vital promotor region of a test gene, they have been shown to selectively repress transcription in a dose-dependent manner. Triplexes have been shown to interfere with the function of sequence-specific DNA binding proteins such as restriction enzymes and transcription factors, both in vitro and in cultured cells.
  • sequence-specific DNA binding proteins such as restriction enzymes and transcription factors
  • the object of the present invention is a synthetic triplex forming oligonucleotide which specifically inhibits steroid hormone dependent transcription.
  • a further object of the present invention is a method of regulating steroid hormone dependent transcription in a cell by the administration of a triplex forming oligonucleotide to the cell.
  • a synthetic triplex forming oligonucleotide comprising: a nucleotide sequence including G and T of about at least 20 nucleotides long, wherein said nucleotide sequence is capable of binding to a DNA duplex target in a sequence coding for a steroid hormone receptor to form a triple helix; and wherein the formation of said triple helix inhibits steroid hormone dependent transcription.
  • a method for regulating steroid hormone dependent transcription in a cell comprises the administration of a triplex forming oligonucleotide to a cell.
  • the triplex forming oligonucleotide specifically inhibits steroid hormone dependent transcription.
  • a method of treating a cell in a pathophysiological state, wherein said treatment comprises contacting the cell with a synthetic triplex forming oligonucleotide to inhibit steroid hormone dependent transcription, said oligonucleotide capable of forming a triple helix with a target sequence in a duplex DNA coding for a steroid hormone receptor.
  • a synthetic oligonucleotide comprising: a nucleotide sequence including G and T of about at least 20 nucleotides long, said nucleotide sequence is capable of binding to a DNA duplex target to form a triple helix; said target being a sequence that binds to a steroid hormone receptor and wherein the formation of said triple helix inhibits activation of the target gene by the steroid hormone receptor.
  • the present invention in still yet another embodiment, also provides a method for regulating steroid hormone receptor target gene activation of a cell.
  • This method comprises the administration of a pharmaceutically effective dose of a triplex forming oligonucleotide to the cell.
  • the oligonucleotide is capable of forming a triple helix with the target gene sequence that binds to a steroid hormone receptor.
  • Figure 1 shows the progesterone-responsive target sequence and the sequence of the triplex-forming oligonucleotides.
  • the double- stranded DNA sequence targeted for triplex formation is shown at the top, SEQ. I.D. No. 9. It contains two well-characterized progesterone response elements (PREs, half-sites indicated by arrows) separated by a linker with high purine/pyrimidine asymmetry.
  • the PRE2ap triplex-forming oligonucleotide (SEQ. I.D. No. 9) was designed to bind to the duplex target as shown. Orientations of DNA strands are indicated at their ends.
  • Two control oligonucleotides are shown: PRE2p (SEQ. I.D. No. 10) and HIV CTR (SEQ. I.D. No. 11).
  • Figure 2 shows- a schematic of the human progesterone receptor gene and TFO target sites for interruption of progesterone receptor function.
  • FIG. 3 shows the PRE2ap TFO forms triplexes with high affinity on PRE-containing targets. Band shift analyses were performed with the radiolabeled double-stranded target SEQ. I.D. No. 9.
  • Figure 4 shows that the PRE2ap TFO binds its PRE- containing target with high specificity.
  • Figure 5 shows that triplex formation inhibits progesterone receptor binding to PREs.
  • FIG. 6 shows that PRE2ap TFO specifically inhibits progesterone receptor-dependent transcription.
  • TFO or "triplex forming oligonucleotide” as used herein refers to the synthetic oligonucleotides of the present invention which are capable of forming a triplex helix by binding in the major groove with a duplex DNA structure.
  • These TFOs can be referred to as the "Hogan” type and are prepared by the "Hogan” technique.
  • This method comprises steps of scanning genomic duplex DNA and identifying nucleotide target sequences of greater than about 20 nucleotides having either about at least 65% purine bases or about at least 65% pyrimidine bases; and synthesizing the synthetic oligonucleotide complementary to the identified target sequence, the synthetic oligonucleotide having a G when the complementary location in the DNA duplex has a GC base pair, having a T when the complementary location of the DNA duplex has an AT base pair.
  • the synthetic oligonucleotide can be selected from the group consisting of an oligonucleotide oriented 5' to 3' and binding parallel to the about at least 65% purine strand, or an oligonucleotide oriented 3' to 5' and binding anti-parallel to the about at least 65% purine strand.
  • the synthetic oligonucleotide for forming a colinear triplex with a target sequence in a duplex DNA when the target sequence is either about at least 65% purine bases or about at least 65% pyrimidine basis comprising, a nucleotide sequence of at least about 20 nucleotides; the nucleotide sequence including G and T, wherein G is used when the complementary location and duplex DNA has a GC base pair and T is used when the complementary location in the duplex DNA is an AT base pair; and the sequence selected from the group consisting of an oligonucleotide oriented 5' to 3' and binding parallel to the about at least 65% purine strand of the duplex DNA target sequence, and an oligonucleotide oriented 3' to 5' and binding anti-parallel to the about at least 65% purine strand in the duplex DNA target sequence.
  • major groove refers to one of the grooves along the outer surface of the double-stranded DNA helix which is formed because the sugar-phosphate backbone of the duplex DNA extends further from the axis than the bases.
  • the major groove is important for binding of regulatory molecules to specific DNA sequences.
  • oligonucleotides as used herein is defined as a molecule comprising two or more deoxyribonucleotides or ribonucleotides, preferably more than 10. The exact size depends on many factors including the specificity and binding affinity.
  • bases herein, the term includes both deoxyribonucleic acids and ribonucleic acids.
  • A refers to adenine as well as its deoxyribose derivatives
  • T refers to thymine
  • U refers to uridine
  • G refers to guanine as well as its deoxyribose derivative
  • C refers to cytosine as well as its deoxyribose derivative
  • F or “N” in sequences refer to guanosine analogues
  • X refers to xanthine as well as its deoxyribose derivative
  • I refers to inosine.
  • steroid hormone receptor superfamily as used herein is defined as a group of proteins some of which are known steroid hormone receptors,with related primary sequences that have widely diverse physiological actions. Steroid hormones interact with intracellular receptors. Examples include estrogen, progesterone and thyroid hormone receptors.
  • diad symmetry as used herein is defined as a palindromic sequence in which the axis of symmetry separates a sequence in a double stranded DNA that has two copies of an identical sequence present in reverse orientation.
  • steroid hormone response element is a synthetic or naturally occurring DNA sequence which, when placed into a heterologous promoter can confer steroid hormone responsiveness to that promoter in the presence of an activated steroid hormone receptor.
  • steroid hormone receptor-dependent transcription as used herein is defined as production of RNA in response to steroid hormones, as mediated by specific steroid hormone receptors.
  • G-free cassette as used herein is defined as a
  • orphan receptor as used herein is defined as member of the steroid receptor superfamily for which no liganda (hormone) has yet been identified.
  • the present invention provides a synthetic triplex forming oligonucleotide comprising: a nucleotide sequence including G and T of about at least 20 nucleotides long, wherein said nucleotide sequence is capable of binding to a DNA duplex target in a sequence coding for a steroid hormone receptor to form a triple helix; and wherein ihe formation of said triple helix inhibits steroid hormone dependent transcription.
  • the present invention also provides a synthetic oligonucleotide comprising: a nucleotide sequence including G and T of about at least 20 nucleotides long, said nucleotide sequence is capable of binding to a DNA duplex target to form a triple helix; said target being a sequence that binds to a steroid hormone receptor and wherein the formation of said triple helix inhibits activation of the target gene by the steroid hormone receptor.
  • Representative examples of triplex forming oligonucleotides of the present invention are shown in SEQ. I.D. Nos. 1, 3, 5, 7, 9, 13, 15 and 17.
  • steroid hormone receptors from the steroid hormone receptor superfamily include estrogen, progesterone, glucocorticoid, mineralocorticoid, androgen, thyroid hormone, retinoic acid, retinoid X,
  • the triplex forming oligonucleotides based on the present invention may bind to any target sequence within the duplex DNA that codes for a steroid hormone receptor so as to inhibit or specifically depress steroid hormone dependent transcription.
  • the target sequence includes the sequence coding for the steroid hormone receptor protein and also the upstream and downstream regulatory elements.
  • the triplex forming oligonucleotides of the present invention may also bind to any target sequence within duplex DNA that is a steroid hormone receptor target gene sequence. It is well known in this art that the steroid hormone receptor complex binds to a target gene sequence with subsequent activation of the target gene.
  • the triplex forming oligonucleotides of the present invention can inhibit activation of the target gene by binding to the steroid hormone receptor target gene sequence.
  • the target sequence within the duplex DNA to which the triplex forming oligonucleotide may be a cis-element, such as a steroid hormone responsive element, or a transcription start site, an elongation region, or a regulatory element.
  • the triplex forming oligonucleotides of the present invention may bind to such regulatory elements as a TATA box, CAAT box, an NF-1 binding site, or steroid hormone response elements.
  • the triplex forming oligonucleotides of the present invention may be administered to any individual. An individual is defined as a human or any animal. Accordingly, the present invention also encompasses pharmaceutical compositions containing the triplex forming oligonucleotides of the present invention.
  • compositions would also suitably contain a pharmaceutically acceptable vehicle, e.g., an oil-based vehicle, water or saline.
  • a pharmaceutically acceptable vehicle e.g., an oil-based vehicle, water or saline.
  • These pharmaceutical compositions of the present invention may be administered in a variety of forms and administration routes well known to those with skill in this art. It is further contemplated that a single dose composition of the present invention may have therapeutic use.
  • the present invention also provides a method for the regulation of steroid hormone dependent transcription within a cell.
  • This method comprises administration of a triplex forming oligonucleotide of the present invention to the cell.
  • the oligonucleotide is capable of forming a triple helix with a target sequence in duplex DNA coding for a steroid hormone receptor. It is specifically contemplated that these methods of treating a cell may occur in vitro or in vivo.
  • the present invention also provides a method for regulating steroid hormone receptor target gene activation in a cell comprising administration of a pharmaceutically effective dose of a triplex forming oligonucleotide to said cell, said oligonucleotide is capable of forming a triple helix with a sequence that binds to a steroid hormone receptor.
  • these methods of regulating gene transcription of a cell may be used to control any steroid hormone receptor transcription or target gene activation.
  • Representative steroid hormone receptors that control gene transcription are described above.
  • the steroid hormone dependent transcription or activation of target gene is promoted by progesterone.
  • a method of treating a cell in a pathophysiological state comprising contacting the cell with a synthetic triplex forming oligonucleotide to inhibit steroid hormone dependent transcription, said oligonucleotide capable of forming a triple helix with a target sequence in a duplex DNA coding for a steroid hormone receptor.
  • any cell in a pathophysiological state wherein depression of steroid hormone dependent transcription is desirable would be an appropriate target for the methods of the present invention.
  • the cellular pathophysiological states are selected from cell proliferative disease, pregnancy and endometriosis.
  • the pathophysiological states are breast cancer, cervical cancer and prostate cancer.
  • Oligonucleotides were synthesized with standard DNA bases according to the method of Durland, et al., Biochemistry 30:9246-9255
  • the target site ( Figure 1) was synthesized as two oligonucleotides with Bgl II and BamHI sites on their 5'ends. After annealing, these were cloned according to ie method of Klein-Hitpass, et al., Cell 60:247-257 (1990), into the Bgl II site of pLovTATA in front of a 400 base pair G-free cassette to construct the pPREP ov template. All TFOs and controls were modified to contain a 3'propy. amine group according to the method of Durland, et al.
  • the target was radiolabeled to high specific activity on the purine-rich strand by EcoRI cleavage of the pPREPLov plasmid and filling in with Klenow and 32 P-dATP and -TTP. After Sac I digestion, the 89 base pair site was gel purified and used in both band shift and footprint analyses.
  • Band-shift assays were conducted as described in Durland, et al., in 20 mM Mg ++ at 37° C. Double-stranded oligonucleotides corresponding to the progesterone receptor element were labeled by DNA polymerase filling-in at the 3' end. These oligonucleotides were then incubated with a TFO and the resulting complex was then separated in polyacrylamide gel electrophoresis. The radiolabeled double stranded DNA target was incubated with increasing amounts of specific and control triplex forming oligonucleotides.
  • Electrophoretic migration of the double stranded target sequence down the acrylamide gel is retarded by the binding of the TFO, but is unaffected by control oligonucleotide.
  • the affinity of the triplex forming oligonucleotide for the target site is then determined by the TFO concentration required for triplex formation. DNase I footprinting was done exactly as described in
  • the target of oligonucleotides was annealed and labeled with Klenow and 32 P-dGTP and -dATP. After gel purification, the target was incubated with TFOs at four times higher than final concentration in 20 mM HEPES, pH 7.9, 24 mM MgCl 2 , and 4 mM spermidine.
  • sheared herring sperm DNA 50/ ⁇ g/ml
  • KC1 + NaCl 45 mM
  • HEPES pH 7.9 (20 mM)
  • dithiothreitol 2 mM
  • MgCLj 6 mM
  • spermidine 1 mM
  • chicken progesterone receptor 50 nM
  • the pPREPLov construct was used as a test template in a cell-free transcription assay modified from that described in Klein-Hitpass, et al.. Cell 60:247-257 (1990).
  • the pPREPLov (100 ng) and AdML 200 control (25 ng) templates were preincubated in 4 ⁇ l with TFOs at concentrations five times higher than final in 12.5 mM HEPES, pH 7.9, 12.5 mM MgCl 2 , and 6.25 mM spermidine at 20° C for 30 minutes.
  • a reaction mix containing buffers, nucleotides (including 32 P-UTP and 3' O-methyl GTP), Tl RNfcse, and herring sperm DNA 750 ng/reaction
  • Either progesterone receptor (one pmole) or BSA was added at that time.
  • fractions of HeLa nuclear extract (DE175, DE500, and S300-II) were added to start transcription.
  • Double-stranded DNA target sites for triplex-helix formation of the Hogan type are presently limited to sequences with segregation of purine and pyrimidine residues to opposite strands.
  • a high affinity TFO target site was engineered based on a well characterized sequence containing tandemly repeated PRE elements of the tyrosine aminotransferase gene. Only the sequence between the PREs was altered to create a purine-rich linker.
  • the new sequence was cloned into the pLov G-free reporter construct, 20 base pairs upstream of a TATA box, in vitro transcription of the G-free reporter responded to progesterone receptor just as did the original PRE-containing construct.
  • the "PRE2ap” TFO (SEQ. I.D. No. 9) and the TFOs designed to inhibit steroid hormone dependent transcription at the level of the steroid hormone receptor gene were designed according to the method described in copending U.S. patent application Serial No. 07/453,532 and EPO application No. 90901460.7, published on October 9, 1991 under No. 0,449,972. That is, for every A:T base pair in DNA duplex, the PRE2ap TFO contained a "T" and for each G:C base pair in DNA duplex the TFO containing a G, a "G" ( Figure 1).
  • TFOs of this kind occupy the major groove of the target double-stranded DNA helix.
  • the region of the target protected from DNase I digestion by the PRE2ap TFO overlaps more than one half-site of each PRE
  • progesterone receptor Since progesterone receptor has been shown to bind as dimers to each PRE, alteration of one half-site interfere with binding to the entire PRE. In addition, binding of progesterone receptor dimers to neighboring PREs is cooperative, so inhibition of binding at one site will also reduce binding at the other.
  • TFO target sites are shown relative to the rest of the gene sequences. Untranslated sequences are indicated by thin lines, while translated sequences are shown as horizontal boxes. In the PR translation product, DNA-binding and ligand binding domains are indicated by diagonal lines and stippling, respectively. Transcription start sites are indicated by the heavy arrows. Cis-elements indicated are binding sites for NF-l/CTF (open oval); SPI (square); and estrogen receptor (triangle).
  • the steroid hormone receptor genes themselves may be inhibited by TFOs.
  • transcription of the progesterone receptor gene could be interrupted at several sites by specific TFOs ( Figure 2).
  • the sites include cis-elements (A, C) transcription start site (B) and elongation regions (C-G).
  • the "G" oligonucleotide would produce a truncated progesterone receptor mRNA that, when translated, would produce a truncated receptor. This receptor could exert a dominant negative effect by dimerizing with wild-type receptors and rendering them inactive by the inability of the truncated receptor to be activated.
  • TFOs of the present invention include a TFO directed against the promoter region of the progesterone receptor gene (SEQ I.D.
  • TFO directed against the transcription start site of the progesterone receptor gene
  • SEQ. I.D. No. 13 TFOs directed against the 5' untranslated region of the progesterone receptor gene
  • SEQ. I.D. Nos. 5 & 7 TFOs directed against the translated region of the progesterone receptor gene
  • the specificity and extent of binding of the PRE2ap TFO to its target sequence was analyzed by DNase I footprinting.
  • the radiolabeled probe an 89 base pair fragment of pPRELov containing the target sequence in Figure 1, was incubated with the PRE2ap TFO or the PRE2p control oligonucleotide, as indicated above the lanes.
  • This autoradiogram shows fragments generated by DNase I digestion of the entire probe except at those regions protected by the binding of the PRE2ap TFO.
  • the triplex extends into both PRE sequences, indicated at right. Alignment of the PRE2ap TFO with the target is indicated by the arrow at right, with "3'A" indicating a
  • the PRE2p control oligonucleotide shows minimal binding to the duplex probe when compared to the "No Oligo" DNase I digestion control lane. Integrity of the duplex binding site is demonstrated at left by the "No DNase” mock reaction.
  • the specific PRE2ap TFO was bound to the target sequence, which was radiolabeled on the 3' end of the purine-rich strand. Limited DNase I digestion yielded a series of fragments arising from cleavage sites present all along the target except at those regions protected by the bound
  • Progesterone receptors bind to the major groove of the double-stranded PRE site. Since the specific TFO bound half of each
  • control oligonucleotide did not affect binding of the receptor. Because progesterone receptor binding was almost completely blocked, triplex formation was most likely complete on the target during the binding incubation. Triplex formation directly correlated to inhibition of progesterone receptor binding. Thus, the PRE2ap TFO competes with progesterone receptors for binding to the PREs.
  • Panel A shows that the in vitro transcription reactions contained G-free cassette templates driven by the PRE-containing TFO target in Figure 1 (filled arrowhead) and control adenovirus major late promotor ("AdML").
  • AdML adenovirus major late promotor
  • cPR chicken progesterone receptor
  • the PRE2ap TFO had minimal effects on AdML transcription (lower bands).
  • the HIV CTR control oligonucleotide did not significantly affect transcription of either template at concentrations as high as 200 nM.
  • Panel B shows that the assay shown in A and an identical one were quantitated on a laser densitometer. Values for progesterone receptor-dependent transcription were normalized to each AdML signal and basal (without cPR) transcription was subtracted.
  • the original template was minimally altered to contain the purine-rich sequence between the two PREs (Figure 1) in front of the G-free cassette. Transcription of this new template was strongly induced by added progesterone receptor in the presence of HeLa cell extract components ( Figure 6A).
  • the adenovirus major late promotor linked to a shorter G-free cassette was used as an internal control in all assays.
  • the TFO was allowed to bind to the template to form a triplex.
  • a final concentration of 25 nM PRE2ap significantly reduced progesterone receptor-dependent transcription while adenovirus control transcription was unaffected (Figure 6A).
  • progesterone receptor-dependent transcription was inhibited to below basal levels while the adenoviral promotor was only minimally affected.
  • Control TFO at a concentration of 200 nM, showed minimal inhibition of test template transcription.
  • the control oligonucleotide reduced test template transcription by less than 40% .
  • the PRE2ap inhibits progesterone receptor-dependent transcription by displacing receptor binding to PREs with potency and specificity.
  • the PRE2ap TFO inhibits only the target promotor, not the AdML control. This should be true even if another steroid hormone responsive (including progesterone responsive) gene is tested since much of the oligonucleotide affinity for the target is dictated by sequences flanking the progesterone-responsive elements. So this TFO inhibits only some progesterone receptor effects.
  • MOLECULE TYPE DNA (genomic)
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • MOLECULE TYPE DNA (genomic)
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • MOLECULE TYPE DNA (genomic)
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • SEQUENCE DESCRIPTION SEQ ID NO:5: GGTTGGTGGT GGTGGTTTTT GTTT 24
  • MOLECULE TYPE DNA (genomic)
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • MOLECULE TYPE DNA (genomic)
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • MOLECULE TYPE DNA (genomic)
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • MOLECULE TYPE DNA (genomic)
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • MOLECULE TYPE DNA (genomic)
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • MOLECULE TYPE DNA (genomic)
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • MOLECULE TYPE DNA (genomic)
  • HYPOTHETICAL NO
  • ANTI- SENSE NO
  • MOLECULE TYPE DNA (genomic)
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • MOLECULE TYPE DNA (genomic)
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • MOLECULE TYPE DNA (genomic)
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • MOLECULE TYPE DNA (genomic)
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • MOLECULE TYPE DNA (genomic)
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • SEQUENCE DESCRIPTION SEQ ID NO:18: GGAGACGGGG GAGGAGAAAJ GGGGAGTCCA GTCGTCATGA CT 42

Abstract

La présente invention concerne de nouveaux oligonucléotides formant des hélices triples, ainsi que leurs procédés d'utilisation. Plus spécifiquement, la présente invention concerne de nouveaux oligonucléotides formant des hélices triples qui inhibent, de façon spécifique, l'activation du gène cible induite par hormones stéroïdes, ainsi que la transcription du récepteur d'hormones stéroïdes. L'invention concerne en outre un procédé de régulation de la transcription d'une cellule dépendant des hormones stéroïdes. Un procédé permettant de traiter une cellule se trouvant dans un état pathophysiologique, par exemple une cellule cancéreuse est également décrit.
PCT/US1993/008329 1992-09-04 1993-09-02 Nouveaux oligonucleotides formant des helices triples, et leurs procedes d'utilisation WO1994005268A1 (fr)

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CHEMICAL REVIEWS, Volume 90, No. 4, issued June 1990, E. UHLMANN et al., "Antisense Oligonucleotides: A New Therapeutic Principle", pages 544-584. *
CONTRACEPTION, Volume 41, No. 3, issued 1990, R. SITRUK-WARE et al., "The use of the Antiprogestin RU-486 Mifepristone as an Abortifacient in Early Pregnancy - Clinical and Pathological Findings; Predictive Factors for Efficacy", pages 221-243. *
MOLECULAR AND CELULAR BIOLOGY, Volume 11, No. 3, issued March 1991, M.-C. PEPIN et al., "Decreased Glucocorticoid Receptor Activity Following Glucocorticoid Receptor Antisense RNA Gene Fragment Transfection", pages 1647-1653. *
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NUCLEIC ACIDS RESEARCH, Volume 19, No. 12, issued June 1991, F. ORSON et al., "Oligonucleotide Inhibition of IL2Ralpha mRNA Transcription by Promoter Region Collinear Triplex Formation in Lymphocytes", pages 3435-3441. *
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES, Volume 88, issued September 1991, E. POSTEL et al., "Evidence that a Triplex-Forming Oligodeoxyribonucleotide Binds to the C-Myc Promoter in HeLa Cells, Thereby Reducing C-Myc mRNA Levels", pages 8227-8231. *
THE EMBO JOURNAL, Volume 9, No. 5, issued 1990, P. KASTNER et al., "Two Distinct Estrogen-Regulated Promoters Generate Transcripts Encoding the Two Functionally Different Human Progesterone Receptor Forms A and B", pages 1603-1614. *
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Cited By (7)

* Cited by examiner, † Cited by third party
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US6441130B1 (en) 1991-05-24 2002-08-27 Isis Pharmaceuticals, Inc. Linked peptide nucleic acids
US6451968B1 (en) 1991-05-24 2002-09-17 Isis Pharmaceuticals, Inc. Peptide nucleic acids
WO1997011170A1 (fr) * 1995-09-20 1997-03-27 Worcester Foundation For Biomedical Research Chimiotherapie par oligonucleotides antisens de l'hypertrophie ou du cancer de la prostate
US6153596A (en) * 1996-12-18 2000-11-28 Emory University Polycationic oligomers
US6440954B1 (en) 1997-04-18 2002-08-27 President And Fellows Of Harvard College Inhibition of vascular smooth muscle cell proliferation
DE19824230A1 (de) * 1998-05-29 1999-12-02 Starzinski Powitz Anna Neues Endometriose-assoziiertes Gen
WO2012167011A1 (fr) 2011-06-02 2012-12-06 Massachusetts Institute Of Technology Forme intermédiaire de réplication du génome hybride arndb/adn des cellules souches métacaryotes

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