EP0449972A1 - Acide nucleique a triple brin et procedes d'utilisation - Google Patents

Acide nucleique a triple brin et procedes d'utilisation

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Publication number
EP0449972A1
EP0449972A1 EP90901460A EP90901460A EP0449972A1 EP 0449972 A1 EP0449972 A1 EP 0449972A1 EP 90901460 A EP90901460 A EP 90901460A EP 90901460 A EP90901460 A EP 90901460A EP 0449972 A1 EP0449972 A1 EP 0449972A1
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EP
European Patent Office
Prior art keywords
synthetic oligonucleotide
group
binding
oligonucleotide
dna
Prior art date
Legal status (The legal status 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 status listed.)
Pending
Application number
EP90901460A
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German (de)
English (en)
Other versions
EP0449972A4 (en
Inventor
Michael Edward Hogan
Donald Joseph Kessler
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Baylor College of Medicine
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Baylor College of Medicine
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Publication of EP0449972A1 publication Critical patent/EP0449972A1/fr
Publication of EP0449972A4 publication Critical patent/EP0449972A4/en
Pending legal-status Critical Current

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Definitions

  • the present invention relates generally to a method for making synthetic oligonucleotides which bind to the major groove of a duplex DNA to form a colinear triplex. It also relates to synthetic oligonucleotides which bind to the purine strand of a DNA duplex. It further relates to a method of regulating and inhibiting cellular growth by administering a synthetic oligonucleotide which is capable of binding to a DNA duplex to form a colinear triplex.
  • the polydT-polydA-polydT triplex is stabilized by T-A Hoogstein base pairing between A in the duplex and the third strand of polydT. That interaction necessarily places the third strand, called a ligand, within the major groove of the underlying duplex.
  • the binding site in the major groove is also referred to as the target sequence.
  • alpha deoxyribonucleotide units of DNA are inherently reversed with respect to the natural beta subunits, an antiparallel triplex formed by alpha oligonucleotides necessarily follows from the observation of parallel triplex formation by the natural beta oligonucleotides.
  • alpha deoxyribo oligonucleotides form parallel rather than antiparallel Watson-Crick helices with a complementary strand of the beta DNA isomer.
  • the present invention provides a new method for designing synthetic oligonucleotides which will bind tightly and specifically to any duplex DNA target.
  • the method can be used to design synthetic oligonucleotides which can be used as a class of drug molecules to selectively manipulate the expression of individual genes.
  • the object of the present invention is a method for designing synthetic obligonucleotides which bind to duplex DNA.
  • a further object of the present invention is a method for making synthetic obliogonucleotides which form triplexes with DNA.
  • a further object of the present invention is a provision of a synthetic oligonucleotide which inhibits the growth of a pathogen.
  • An additional object of the present invention is a method for altering the structural protein content of epidermal tissue for the treatment of aging and blood clotting.
  • a further object of the present invention is a method of inhibiting gene expression by permanently altering the DNA sequence.
  • a method for making a synthetic oligonucleotide which binds to a target sequence in duplex DNA forming a colinear triplex by binding to the major groove comprising the 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 said synthetic oligonucleotide complementary to said identified target sequence, said 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.
  • a further aspect of the present invention is the synthetic oligonucleotide for forming a colinear triplex with a target sequence in a duplex DNA when said 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; said 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 said 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.
  • the synthetic oligonucleotide can have at least one T replaced by X, I, and halogenated derivatives of X and I. Furthermore, at least one G can be replaced with halogenated derivatives of G.
  • Additional embodiments include substitutions on the synthetic oligonucleotide.
  • the base can be substituted at the 2' furanose position with a non-charged bulky group and the backbone of the synthetic oligonucleotide can be a phosphodiester anologue which is not readily hydrolyzed by cellular nucleases.
  • a linker can be affixed at the 3' and/or 5' terminus of the synthetic oligonucleotide. This linker provides a method for attaching modifying groups to the oligonucleotide.
  • the modifying groups can be intercalators, groove-binding molecules, cationic amines and cationic polypeptides.
  • Another aspect of the present invention is a method of inhibiting the growth of cells comprising the step of administering synthetic oligonucleotides in sufficient quantity for cellular uptake and binding to the target sequence, wherein said target sequence is positioned within the DNA domain adjacent to the RNA transcription origin.
  • This procedure can be used to inhibit the growth of cancer cells and pathogens. In one preferred embodiment this procedure is used to inhibit HIV-I virus by binding a synthetic oligonucleotide to the viral LTR region.
  • Another aspect of the present invention is a method of altering the relative proportions of the structural protein content of epidermal tissue by administering a synthetic oligonucleotide in sufficient quantity for cellular uptake and binding to target sequences for collagen genes.
  • Fig. 1A shows the surface morphology of a colinear triplex. It is a computer generated rendering of the structure of a duplex DNA target site and presents in both the canonical B and A helix form.
  • the target Upon binding of an oligonucleotide ligand, the target undergoes a transition from the B to the A form, which creates an increase in the depth of the major helix groove (M) .
  • M major helix groove
  • the oligonucleotide wraps about the A form helix target, occupying the major groove. The groove binding has been emphasized by presenting the bound oligonucleotide as a ribbon-like abstraction.
  • Fig. IB shows the strand orientation in a colinear triplex.
  • the oligonucleotide ligand binds to the duplex target, in the parallel orientation relative to the orienting (more purine rich) strand.
  • Fig 2 shows the pattern of oligonucleotide hydrogen bonding with the duplex target: G to GC sites, T to AT sites.
  • 2A is a computer simulated rendering of the preferred pattern of hydrogen bonding between G in the ligand and G in the GC base pair at the corresponding site within the orienting strand of the duplex target.
  • 2B is an equivalent simulation of T binding to the A of an AT base pair at its corresponding site within the orienting strand of the duplex target.
  • the T-AT association is identical to classical "Hoogsteen base pairing", whereas the G-GC association is essentially the quanine counterpart thereof and involves N3 to 06 bonding.
  • Solid wedges define the site at which such a crossection through a triplex is affixed to the corresponding crossection above it.
  • Open wedges define the site at which such a crossection through a triplex is affixed to the corresponding crossection below.
  • the connectivity defined by the two bonding schemes is nearly identical. It is also important to recognize that the favored pattern of bond formation between G and GC or T and AT (arrows) cannot be mimicked by any other pattern of base-base association at neutral pH (C can mimic G in acid conditions) .
  • 2C and 2D are corresponding bonding patterns which result when the G of a GC base pair or A of an AT pair occurs across from the orienting strand of the target duplex.
  • the rules of oligonucleotide sequence selectivity are the same (i.e., G at a GC pair, T at an AT pair) however, G bonding occurs N3 to N9 and T bonds in the "reverse Hoogsteen" way, thereby both retain the overall parallel orientation of the bound ligand and the orienting strand of the target.
  • Fig. 3 shows one method of improving the pattern of oligonucleotide hydrogen bonding with the duplex target: xanthine binding to AT sites.
  • the computer generated simulation in Fig. 3 is as in Fig. 2, except that the effect of substituting xanthine (X) for T is presented.
  • X xanthine
  • X is nearly identical to the G residues which might flank it in an oligonucleotide ligand, with respect to base size and shape and with respect to the orientation of its phosphodiester component within the oligonucleotide binding site.
  • Modeling predicts that such enhancement of oligonucleotide continuity will enhance the binding affinity and site specificity of all oligonucleotides in which T is replaced by X.
  • Fig. 4 displays the family of altered phosphodiester linkages compatible with colinear triplex formation.
  • Fig. 5 shows formation of hybrid olionucleotides by means of coupling through a 5' amine linkage.
  • a hexylamine linkage is described.
  • This linkage can be affixed as the last residue of an oligonucleotide by employing the same phosphoramidite chemistry used to polymerize the DNA bases.
  • groups which selectively react with a primary alkyl amine can be added. These groups include the isothiocyanate derivative of eosin (EITC) or 9 amino acridine (AIT) , or any number of other small molocules.
  • EITC isothiocyanate derivative of eosin
  • AIT 9 amino acridine
  • Fig. 6 shows dose dependent inhibition of HIV-1 mRNA by Oligonucleotide mediated DNA triplexes.
  • U937/HIV-1 cells ATCC CRL 1593, American Type Culture Collection, Rockville, MD
  • HTLV-IIIB prototype strain of HIV-1 infected with the HTLV-IIIB prototype strain of HIV-1 and cultured under conditions where >90% of the cells remained viable and contained HIV-1 mRNA as shown by in situ hybridization with the 35 S-labeled probe for the LTR of HIV-1, (NEP 200, DuPont, Wilmington, DE)] were incubated with each oligonucleotide at 0, 2, 6, 10, and 20 uM concentrations. Oligonucleotide was added to the culture supernatants at -li ⁇
  • RNAzol (Cinna/Biotecx Laboratories International, Inc., Friendswood, TX) .
  • Serial 2-fold dilutions were made from each RNA preparation (starting at 2.5 ug RNA) and equal amounts were applied to duplicate nylon membranes using a slot blot apparatus (Biorad) .
  • One blot was probed with the radiolabeled EcoRl-Hhal env fragment from the HIV-1 containing plasmid pARV-7/2, while the other was probed with radiolabeled cDNA for ⁇ -actin.
  • the resulting autoradiographs were then analyzed by densito etry.
  • the density units expressed on the ordi ate express the ratio of (env-probe density) / (actin-probe density).
  • ⁇ represent HIV29par
  • D represent HIV31 anti
  • D represent random HIV29 isomer.
  • Fig. 7 shows the persistence of the effect of oligonucleotides on HIV infected H9 T cells.
  • HIV-1 infected U937 cells were cultured for 12 to 72 hrs . after the last addition of HIV31anti. The oligonucleotide was added at the initiation of the culture and at 2 hrs. thereafter to maintain a final concentration of lO ⁇ M. Cells were harvested at the indicated time points thereafter. Total cellular RNA was harvested and applied to duplicate nylon membranes in serial dilution with a slot blot apparatus. One replicate was probed with the HIV-1 env cDNA and the other with the cDNA for ⁇ -actin.
  • the density units are expressed as the ratio of env to ⁇ -actin densitometry readings.
  • ⁇ represent HIV31 anti and O represent controls.
  • Fig. 8 shows inhibition of viral mRNA by HIV29par in infected H9 cells.
  • the densitometric analysis shows a decrease in specific viral message.
  • H9 cells, infected with HTLV IIIB, were treated with oligomer (5 ⁇ M) every two hours. At four and twelve hours the cells were harvested, washed with PBS, and the total cellular RNA was extracted. The hatched bars represent oligomer treatment and unhatched bars represent controls.
  • synthetic oligonucleotides as used herein is defined as a molecule comprised of two or more deoxyribonucleotides or ribonucleotides, preferably more than ten. Its exact size will depend on many factors, including its specificity and binding affinity.
  • the term includes both deoxyribonucleic acids and ribonucleic acids.
  • A refers to adenine as well as its deoxyribose derivatives
  • T refers to thymine as well as its deoxyribose derivative
  • G refers to quanine as well as its deoxyribose derivative
  • C refers to cytosine as well as its deoxyribose derivative
  • X refers to xanthine as well as its deoxyribose derivative
  • I refers to inosine.
  • the "major groove' refers to one of the grooves along the outer surface of the DNA helix which is formed because the sugar-phosphate backbone extends further from the axis than the bases do.
  • the major groove is important for binding of regulator molecules to specific DNA sequences.
  • One embodiment of the present invention is a method for making a synthetic oligonucleotide which binds to a target sequence in duplex DNA forming a colinear triplex by binding to the major groove, said method comprising the steps of: scanning genomic duplex DNA and identifying nucleotide target sequences of greater than 20 nucleotides, said target sequences having either about at least 65% purine bases or about at least 65% pyrimidine bases; and synthesizing said synthetic oligonucleotide complementary to said identified target sequence, said 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 in the DNA duplex has an AT base pair.
  • the synthetic oligonucleotide is selected from the group consisting of an oligonucleotide oriented 3' to 5' and binding anti-parallel to the about at least 65% purine strand and an oligonucleotide oriented 5' to 3' and binding parallel to the about at least 65% purine strand.
  • the resulting oligonucleotide can be synthesized in gram quantities by the standard methods of solid phase oligonucleotide synthesis.
  • the site-specific oligonucleotide procedure is divided into three parts:
  • an oligonucleotide length must be chosen. There is a one to one correspondence between oligonucleotide length and target length. For example, a 27 base long oligonucleotide is required to bind to a 27 base pair long duplex DNA target. Under optimal conditions, the stability of the oligonucleotide-duplex DNA interaction generally increases continuously with oligonucleotide length.
  • a DNA oligonucleotide in the range of about 20 to 40 bases is used. Oligonucleotides in this range usually have useful dissociation constants for their specific DNA target. The dissociation constants are in the range of about 10 —9 to 10—8 molar.
  • Oligonucleotides shorter than 20 bases display weaker and less specific binding to the target sequence and are thus less useful.
  • Oligonucleotide binding to duplex DNA is stabilized by binding to the purines in the underlying duplex. Once a DNA target has been identified, the more purine rich strand of the target area is defined as the "orienting" strand of the binding site.
  • An oligonucleotide ligand was designed to bind either parallel or anti-parallel to the orienting strand. The stability of the binding is dependent on the size of the oligonucleotide and the location in the genome. Sometimes the parallel is more stable than the anti-parallel while at other times the reverse is true or they are equally stable.
  • the method of designing a detailed sequence of an oligonucleotide ligand involves placing a T in the oligonucleotide whenever an AT base pair occurs in the duplex target, and placing a G in the oligonucleotide whenever a GC base pair occurs in the duplex target.
  • Another embodiment of the present invention includes a synthetic oligonucleotide for forming a colinear triplex with a target sequence in a duplex DNA when said target sequence is either about at least 65% purine bases or about at least 65% pyrimidine bases, comprising, a nucleotide sequence of at least about 20 nucleotides; said nucleotide sequence including G and T, wherein G is used when the complementary location in the duplex DNA is a GC base pair and T is used when the complementary location in the duplex DNA is an AT base pair; and said sequence selected from the group consisting of an oligonucleotide oriented 3' to 5' and binding anti-parallel to the about at least 65% purine strand in the duplex DNA target sequence and an oligonucleotide oriented 5' to 3' and binding parallel to the about at least 65% purine strand in the duplex DNA target sequence.
  • any duplex DNA sequence of about at least 65% purines can form a stable triplex.
  • duplex DNA sequences which have only purines on the template strand form complexes which in general, are characterized by enhanced stability. If we define n as the number of bases within the template strand which are purine and define (1-n) as the number of pyrimidine bases in the template, then the approximate dissociation constant can be predicted from the following semi-empirical formula:
  • This formula assumes near-physiological conditions in vitro, that is 0.05 M TRIS/HC1, 5mM MgCl 2 , 3mM spermine pH 7.8, 37°C. These conditions constitute the operating standard used in the design process.
  • oligonucleotides are synthesized by the phosphoramidite method, thereby yielding standard deoxyribonucleic acid oligomers.
  • Molecular modeling suggests that substitution of the non-hydrolyzable phosphodiester backbone in the oligonucleotide or elected sites may enhance the stability of the resulting triplex in certain instances.
  • the phosphodiester analogues are more resistant to attack by cellular nucleases.
  • non-hydrolyzable phosphodiester backbones examples include phosphorothioate, phosphoroselenoate, methyl phosphate, phosphotriester and the alpha enantiomer of naturally occurring phosphodiester.
  • the thiophosphate and methyl phosphonate linkages are shown in Fig. 4.
  • Backbone modification provides a practical tool to "fine tune" the stability of oligonucleotide ligands inside a living cell. For example, oligonucleotides containing the natural phosphodiester linkage are degraded over the course of 1-2 hours in eukaryotic cells, while the non-hydrolyzable derivatives appear to be stable indefinitely.
  • Oligonucleotide hybrids provide another method to alter the characteristics of the synthetic oliogonucleotides.
  • Linkers can be attached to the 5* and/or 3' termini of the synthetic oligonucleotide.
  • the linkers which are attached to the 5' terminus are usually selected from the group consisting of a base analogue with a primary amine affixed to the base plane through an alkyl linkage, a base analogue with a sulfhydryl affixed to the base plane through an alkyl linkage, a long chain amine coupled directly to the 5' hydroxyl group of the oligonucleotide and a long chain thiol coupled directly to the 5' hydroxyl group of the oligonucleotide.
  • the linker on the 3* terminus is usually a base analogue with a primary amine affixed to the base plane through an alkyl linkage or a base analogue with a sulfhydryl affixed to the base plane through a alkyl linkage. Affixation of a primary amine linkage to the terminus does not alter oligonucleotide binding to the duplex DNA target.
  • modifying groups can be attached to the synthetic oligonucleotide.
  • the molecules which can attach include intercalators, groove-binding molecules, cationic amines or cationic polypeptides.
  • the modifying group can be selected for its ability to damage DNA.
  • the modifying group could include catalytic oxidants such as the iron-EDTA chelate, nitrogen mustards, alkylators, photochemical crosslinkers such as psoralin, photochemical sensitizers of singlet oxygen such as eosin, methylene blue, acridine orange and 9 amino acridine and reagents of direct photochemical damage such as ethidium and various pyrene derivatives.
  • catalytic oxidants such as the iron-EDTA chelate, nitrogen mustards, alkylators, photochemical crosslinkers such as psoralin, photochemical sensitizers of singlet oxygen such as eosin, methylene blue, acridine orange and 9 amino acridine and reagents of direct photochemical damage such as ethidium and various pyrene derivatives.
  • the modified oligonucleotides can be coupled to any reagent which is specific for a primary amine, for example a succimidate or isothiocyanate moiety (Fig. 5).
  • an "aminolink" coupling is used to affix the intercalating dyestuff 9 acridine isothiocanate to triplex forming oligonucleotides.
  • the duplex binding affinity of the oligonucleotide-dye hybrid is approximately 100-fold greater than the oligonucleotide binding affinity.
  • Other embodiments include affixing eosin isothiocyanate to oligonucleotides. Since eosin isothiocyanate cleaves the DNA helix upon irradiation this hybrid oligonucleotide cuts the helix at its binding site when irradiated. This hybrid-oligonucleotide is useful for identifying the oligonucleotide binding site both in vitro and in vivo and potentially can be used as a therapeutic tool for selective gene target destruction.
  • Photochemical reactivity is also achieved by affixation of psoralin derivatives to oligonucleotides through a 5' linkage.
  • Psoralin binds covalently to DNA after irradiation, and as a consequence is a potent cytotoxic agent.
  • photochemical reactivity, with oligonucleotide sensitivity provides a tool to direct the toxic psoralin lesion to the oligonucleotide target site.
  • Similar oligonucleotide coupling is used to target toxic chemical reactivity to specific DNA sequences.
  • Examples include catalytic oxidants such as transition metal chelates and nucleases.
  • Photochemical reactivity and/or toxic chemical agents can be used to permanently inhibit gene expression.
  • modifications of oligonucleotides alter the rate of cellular uptake of the hybrid oligonucleotide molecules.
  • the uptake process is rapid, but poorly understood.
  • Terminal modification provides a useful procedure to modify cell type specificity, pharmacokinetics, nuclear permeability, and absolute cell uptake rate for oligonucleotide ligands.
  • C. Modified base analogues provide another means of altering the characteristics of the synthetic oligonucleotide. Although a purine rather than a pyrimidine, X is identical to T with respect to its capacity to form hydrogen bonds.
  • substitution of X for T in the above oligonucleotide design procedures results in a modified triplex that is much more stable.
  • the increased stability is due principally to enhanced stacking and to an enhancement of phosphodiester backbone symmetry within the ligand.
  • base substitutions for T are X, I and halogenated X and I.
  • G can be replaced by halogenated G.
  • the 2' furanose position on the base can have a non-charged bulky group substitution.
  • non-charged bulky groups include branched alkyls, sugars and branched sugars. In the preferred embodiment at least one base is substituted.
  • oligonucleotide design will produce ligands with target affinity and specificity which exceeds that of even the most specific antigen-monoclonal antibody interaction.
  • Synthetic oligonucleotides have been designed to the transcription control region of the human c-myc protooncogene, to the regulation sequence of collagen I ⁇ , to bind to the TATA box segment of the chicken alpha actin gene, and to bind to an enhancer sequence within the early gene region of human HIV-I.
  • a further embodiment of the present invention is a method of inhibiting the growth of cells, comprising the step of administering a synthetic oligonucleotide in sufficient amount for cellular uptake and binding to the target sequence, wherein said target sequence is positioned within the DNA domain adjacent to the RNA transcription origin.
  • the synthetic oligonucleotide is as described above in the description of the design process. Uptake into the cells is rapid for these synthetic oligonucleotides and can be altered with the appropriate substitutions and modif cations. Similarly the binding can be altered by appropriate changes to the synthetic oligonucleotide.
  • the inhibition of cell growth can be used in the treatment of cancerous cells. Additions of the specific oligonucleotide will selectively inhibit cell growth.
  • synthetic oligonucleotides to the c-myc gene can be used to inhibit some cancerous cell growth.
  • synthetic oligonucleotide which inhibit c-myc expression include: 3 '-TGGTGTGTGGGTTTTGTGGG GGGTGGGGGGGTTTTTTTTGGGTGGG-5 • and/or 3'-TGTGGTGGGGTGGTTGGGGTGGGTGGGGTGGGTGGG-5' and/or 5'-TTTGGTGTGGGGGTGGGGGTTTTGTTTTTTGT-3' and/or 3'-GGTTGGGGTGGGTGGGGTGGGTGGGGT-5 • and/or 5'-GGTTGGGGTGGGTGGTGGGTGGGGT-3' and fragments and analogues thereof.
  • Another embodiment includes a method of inhibiting the growth of pathogens comprising the step of administering a synthetic oligonucleotide in sufficient amount for cellular uptake and binding to the target sequence, wherein said sequence binds within the nucleic acid domain adjacent the RNA transcription origin.
  • a synthetic oligonucleotide which selectively binds to the viral LTR region.
  • this synthetic oligonucleotide can include 3'-GTTTTTGGGTGTTGTGGGTGTGTGTGTGGTT-5 • and/or
  • An additional embodiment includes a method of manipulating the structural protein content of epidermal tissue comprising the step of administering a synthetic oligonucleotide in sufficient amount for cellular uptake and binding to the target sequence. This includes inhibiting the various enzymes and regulating proteins in skin.
  • the collagen I ⁇ gene synthesis rate can be altered by using 3 • -TGGGTTGGGTGGTGGTGGGGGTGTGGTTTGGTTGTGGGTTTTT-5' and/or 3'-GTGGGTTGGGTGGTGGTGGGGGTGTGGTTTGG-5' and fragments and analogues thereof as the synthetic oligonucleotide.
  • collagenase gene can be inhibited by using 5'GGTTGGGGTTGGTGTGTTTTTTTTTTGTGTGGGTG-3* and/or 5'-TTGTGGTTGTTTTTTTGGTTGTGTGT-3, and fragments and analogues thereof.
  • the synthetic oligonucleotides described in the examples can include any of the substitutions discussed earlier.
  • the backbone, base, linkers and modifying groups can be added. These substitutions will enhance the affinity, the chemical stability, and the cellular uptake properties of the specific oligonucleotide treatments.
  • the c-myc gene In both cancerous and normal cells, the c-myc gene possesses several target sequences within its 5' flanking sequence which satisfy the synthetic oligonucleotide design criteria. In a program of drug development, these target sequences and others are used as templates to direct oligonucleotide design. The purpose of these oligonucleotides is to selectively inhibit c-myc transcription, thereby repressing the uncontrolled growth of tumors with the c-myc lesion.
  • triplex oligonucleotides described here were designed to selectively repress the expression of the c-myc gene in such tumors, thereby slowing tumor growth.
  • the 27 bp target duplex has 74% GC base pairs and 89% ppuurriinnee oonn tthhee oorriieennttiinngg ssttrand.
  • the Kdiss is (6x10 M) for anti-parallel binding.
  • TARGET SEQUENCE BETWEEN TATA BOX AND ACTIVATOR SITE IN A HIGHLY.
  • Both synthetic oligonucleotides A and B specifically interact within the target duplex to inhibit tumor growth, by means of specific repression of c-myc transcription.
  • the specific method of inhibition of oligonucleotide C is unknown.
  • oligonucleotides for other genes involved in human tumors can be similarly designed. The procedure is only limited by the available molecular sequence data.
  • the structural proteins which define the mechanical properties of skin are well known.
  • the molecular structure of the collagen and elastin proteins and their corresponding proteases, collagenase and elastase, have been intensley studied. These proteins are under the control of an elaborate program of regulation, which appears to change during the wound healing process and as a result of the aging process.
  • the molecular structure is sufficiently defined to consider treatments based upon gene-specific intervention into the pattern of structural protein synthesis and/or enzymatic degradation.
  • a program of synthetic oligonucleotide design, based upon manipulation of collagen I synthesis in human skin is described below. By altering the relative protein concentrations the structure and mechanical properties of skin can be altered.
  • the synthetic oligonucleotide can be used as a therapeutic agent to alter the skin aging process or to alter the wound healing process.
  • One skilled in the art will readily recognize that the concepts can be extended to other collagens, to other skin proteins and to their complementary proteases based upon the availability of the necessary genetic data.
  • target sequences in the transcription control region of the human alpha 1(1) collagen gene the likely function of those sites, their position relative to the RNA transcription origin, and the synthetic oligonucleotide sequence designed for collagen specific treatment as shown below. As the molecular genetics of the collagen gene develops, the list of target sequences within the 5' flanking region will be expanded.
  • Synthetic oligonucleotides A and B inhibit type I collagen protein synthesis.
  • the process includes the specific repression of collagen RNA transcription.
  • the method of inhibition of the C synthetic oligonucleotide is not known.
  • the effect on protein synthesis of skin proteins can be seen by adding sufficient amounts of the synthetic oligonucleotide for uptake into cultured human fibroblasts.
  • the D synthetic oligonucleotide inhibits collagenase protein synthesis.
  • the process includes specific repression of collagenase RNA transcription.
  • the E synthetic oligonucleotide causes loss of TPA sensitivity, and a subsequent repression of collagenase syntheses in the presence of promotors such as TPA.
  • This process includes specific repression of collagenase RNA transcription.
  • Synthetic oligonucleotide interaction will cause collagen protein levels in the cell to rise, as collagenase levels fall. The clinical effect of the increase should cause a useful alteration of the mechanical properties of skin. The effects can be seen by adding sufficient amounts of oligonucleotide for cellular uptake to cultured human fibroblasts.
  • the HIV-I virus is known to be the causative agent in human acquired immune deficiency syndrome (AIDS) .
  • the long terminal repeat of the HIV-1 virus is known to possess several DNA segments within the LTR region which are required for transcription initiation in a human T-cell host.
  • the synthetic oligonucleotides selectively repress HIV-1 mRNA synthesis in a human host cell, by means of triplex formation upon target sequences within the viral LTR. Repression of an RNA synthesis results in the reduction of the growth rate of the virus. This could result in the slowing of the infection process or the repression of the transition from latency to virulent growth.
  • Most of the sites within the LTR will comprise target sites for drug (oligonucleotide) intervention. There is no wasted DNA in the small, highly conserved LTR region.
  • target sequences in the transcription control region of the human HIV-1 LTR the likely function of these sites, their position relative to the RNA transcription origin, and the oligonucleotide sequence designed as a HIV-I specific treatment are shown below.
  • the list of target sequences within the LTR and elsewhere will be expanded. In all instances, both the parallel and antiparallel isomers are described. The reason is that, although one or the other will always display the better binding affinity in vitro, the efficacy of each must be tested in vivo to make the final decision.
  • TARGET SITE A segment of the negative HIV1 regulatory domain, with similarity to a homologous domain in interleukin 2 gene.
  • C TARGET SITE: A site near the center of the LTR.
  • the HIV4 par also functions if TG is added to the 3' end to make HIV38 par.
  • HIV29par and HIV31anti were designed as previously described herein. HIV31anti also functions if bases two TG are removed from the 3' end.
  • the relative mobility and DNA footprint analyses of both oligonucleotides show binding with high affinity to target proviral sequences, in. vitro. HIV-1 infected U937 cells, a monocytoid line, were treated with up to 20 ⁇ M with either HIV29par, HIV31anti, or a random isomer of HIV29 with no detectable in vitro affinity for the target sequence.
  • HIV29par the cells were able to rapidly sequester the oligomer in concentrations exceeding that of the media.
  • H9 T cells Treatment every 2 hrs. with 5 ⁇ M effectively suppressed mRNA synthesis in HIV-1 infected H9 T cells at
  • the evidence shows that the oligonucleotides designed to bind within the major groove of the DNA helix, and form triplexes with specific gene sequences in the tar region of the HIV-1 provirus are readily taken up by HIV-1 infected cells and selectively suppress synthesis of HIV-1 mRNA without concomitant suppression of mRNA for ⁇ -actin, which constitutive expressed in these cells. With inhibition of viral MNRA synthesis, translation of virus-encoded proteins is also suppressed. Inhibition of viral mRNA depended on the dose of oligonucleotide added; maximum inhibition occurred at concentrations > lO ⁇ M.
  • oligonucleotides designed to bind to specific sequences in the DNA duplex and form colinear triplex with the targeted sequences provide an efficient and highly specific agent for regulating gene expression, such agents provide a new class of rationally designed chemotherapeutic agents for controlling virus replication and other processes depend upon new mRNA production.
  • the synthetic oligonucleotides in A through E will inhibit HIV-I mRNA synthesis, hence viral growth.
  • the process includes specific repression of RNA transcription from the viral LTR.
  • a representative target sequence in the transcriptions control region of the chicken skeletal alpha actin gene, the function of that site, its position relative to the RNA transcription origin, and the oligonucleotide sequence which would be designed as an actin specific treatment are shown below. As the molecular genetics of the actin gene develops, the list of target sequences within the actin control region will be expanded.
  • This synthetic oligonucleotide molecule inhibits actin protein sythesis, by specific repression of RNA transcription. This inhibition can be assessed in cultured chicken myoblasts. The intact chicken will show a change in the quality of actin and other muscle proteins whose synthesis if strongly coupled to actin expression. The practical result of this change will be an alteration of the properties of chicken meat.
  • the 28bp target is comprised of 54% G+C base pairs and is
  • the APP770 Gene is the precursor protein responsible for production of plaque in Alzheimers disease, A. TARGET SITE: DOWNSTREAM TATA BOX SITE
  • EGFR epidermal growth factor gene
  • TARGET SITE The target domain comprizes the consensus binding sequences for the transcription activating factors API and Spl. Synthetic Oligonucleotides targeted against this will repress GSTpi transcription by means of competition with API and Spl. DNA Duplex Target
  • TARGET SITE An enhancer-like polypurine sequence. A synthetic oligonucleotide targeted against this site will repress GSTpi transcription by means of competition with the enhancer.
  • HMGCoA Reductase is the enzyme which defines the rate limiting step in cholesterol biosynthesis. Its molecular genetics has been studied to understand the control of cholesterol synthesis. The described synthetic oligonucleotides will intervene in the program of cholesterol synthesis by means of modulating the transcription of HMGCoA.
  • TARGET SITE The target is binding site for a repressor protein that appears to mediate end-product inhibition of transcription by cholesterol.
  • the synthetic oligonucleotide is a synthetic repressor of HMGCoA expression, as an agonist of the cellular repressor.
  • TARGET SITE The target is a binding site for protein that appears to activate transcription of HMGCoA.
  • the synthetic oligonucleotide against this site is a synthetic repressor of HMGCoA expression, as an antagonist of the cellular protein which binds to the target.
  • TARGET SITE The target is a binding site for a protein that appears to activiate transcription of HMGCoA by binding to the "TATA box" domain.
  • a TFO against this site is designed to be a synthetic repressor of HMGCoA expression, as an antagonist of the cellular protein which binds to the TATA box target.
  • NGFR Nerve Growth Receptor
  • the NGFR gene encodes a cell surface receptor required for nerve cell proliferation. It is overexpressed in neuroblastoma and melanomas. Triplex oligonucleotides are designed to repress the growth of those cancerous tissues. Activation of the gene would be a precondition of activation of nerve cell regeneration.
  • the mRNA start site is at -122 in this number scheme.
  • A. TARGET SITE Consensus Spl binding site
  • TARGET SITE Consensus SPl binding site.
  • NGFR2anti C.
  • TARGET SITE Domain flanking consensus Spl binding sites.
  • TARGET SITE Domain flanking consensus Spl binding sites.
  • ANTI-PARALLEL SYNTHETIC OLIGONUCLEOTIDE SEQUENCE 3 '-GGGTGGGTTTGGGTGTGGTTGGGTGGGG-5 ' (NGFR4anti)
  • TARGET SITE Consensus Spl binding site.
  • HERPES SIMPLEX VIRUS 1 DNA Polymerase and DNA binding proteins
  • HSV-1 is responsible for a variety of skin lesions and other infections.
  • the triplex oligonucleotide are designed to bind directly to the promotor region of the genes which encode the viral DNA polymerase and DNA binding protein, thereby arresting viral replication. Both genes occur at 0.4 map units and flank the replication origin oriL. Numbering below is in terms of the polypeptide start site for each gene.
  • A. TARGET SITE This site is in the 5' flanking sequence of the DNA polymerase gene.
  • the Angelotti strain has three base changes relative to strain 17.
  • ANTI-PARALLEL SYNTHETIC OLIGONUCLEOTIDE SEQUENCE 5—TTTTTGTGTTGGGGGGGTGGGGTGTTGGGGGGTGTTT-3 ' (HSVPOLlanti)
  • TARGET SITE This site is in the 5" flanking sequence of the DNA binding protein gene for strain 17.
  • HSV-1 is rseponsible for a variety of skin lesions and other infections.
  • the triplex oligonucleotides are designed to bind directly to the two classes of HSV-1 DNA replication origin, thereby arresting viral replication.
  • the first origin (oriL) occurs at 0.4 map units and is in between and immediately adjacent to the HSV-1 DNA polymerase and DNA binding protein genes.
  • the two identical origins of the second type (oriS) occur at 0.82 and 0.97 map units. Numbering below is the terms of position relative to the two fold symmetry axis of each origin.
  • oriL also comprises the 5* flanking domain of the HSV-IDNA polymerase and the HSV-1 major DNA binding protein, these triplex oligonucleotides may also interfere with transctiption of those two genes.
  • the beta globin gene encodes one of the proteins comprising adult hemoglobin. Mutation in this gene is responsible for beta thalassemia and sickle cell anemia. Triplex oligonucleotides targeted to this gene are designed to inhibit the beta globin gene in thallassemics and in patients with sickle cell anemia, to be replaced by the naturally occuring delta protein. Two classes of triplex oligonucleotides TFO are described, which are targeted against the 5' enhancer or the promotor/coding domain. Numbering is relative to the principal mRNA start site.
  • oligonucleotide binding in cells The effects of triplex-forming oligonucleotides are studied in cell culture. Oligonucleotides are administrered to cultured human cell lines, which are then analyzed for oligonucleotide uptake and for a change in the steady-state level of messenger RNA associated with the DNA target. As an example, the methods for the c-myc gene are shown. One skilled in the art will readily be able to generalize to any gene within a cultured cell.
  • HeLa cells grown on a solid support are treated with 32P-labelled oligonucleotide, then incubated as a function of time and concentration.
  • the uptake rate i.e., the time constant to reach a steady state with respect to oligonucleotide uptake.
  • Transcription inhibition is assayed by a variation of the RNase protection assay, which is the standard assay for quantitying steady state mRNA levels in mammalian cells.
  • Total cellular RNA is extrated from oligonucleotide-treated HeLa cells, then hybridized to a uniformly labelled antisense RNA transcript, generated by the action of T7 polymerase on the Smal-PvuII human c-myc frangment in pSPT19.
  • This Smal-PvuII probe is complementary to the first myc exon and sequences which comprise both the PI and P2 transcription start sites of myc.
  • a limit RNasel digest produces either a 0.6 kb duplex (transcription from PI, which is the preferred origin in HeLa cells) or a 0.4 kb duplex (transcription occurs instead from P2, which is used in HeLa cells under conditions of serum starvation) .
  • the size and quantity of the resulting RNase resistant duplexes is then determined by quantitative autoradiography on a 5% acrylamide gel matrix.
  • This assay system can quantify steady-state RNA levels to within 20% accuracy, which is sufficient for the purposes of this analysis .
  • the outcome of these cellular titrations is analyzed in the context of two control experiments. The first is a comparison of the dose response of oligonucleotides which bind selectively to the target gene and the dose response of oligonucleotides which are unrelated. If oligonucleotide-mediated repression of the c-myc transcription is due to site-specific triplex formation in the cell, then an unrelated oligonucleotide will not elicit an affect, over an equivalent concentration range.
  • the second control addresses the gene specificity of the effect.
  • data are always normalized to overall RNA concentation in the cell. As such, changes in the steady state level of the myc transcript are meaningful in their own right.
  • myc-specific oligonucleotide treatment on the steady state levels of the histone 2A (H2A) message in HeLa cells, probing the RNA complement with an H2A antisense RNA, generated from a construct which, as for myc sequences, has been cloned into a RNA expression vector.
  • H2A histone 2A
  • oligonucleotides which bind to the control region of the human c-myc gene selectively repress c-myc transcription in an intact HeLa cell.
  • Preliminary work with other oligonucleotides described in the examples have begun to display similar selectivity.
  • application of these methods is readily generalized to any gene in any cell line and is limited only by the availability of cloned gene constructs, DNA sequence data, and a rudimentary understandinng of the molecular genetics of the gene under investigation. At present, that battery of information is availale for several hundred human genes, and for several thousand genes from other species.
  • oligonucleotides variants such as those with chemical moieties added to the 3' nd 5' terminus, oligonucleotides with an altered phosphodiester backbone or those with bases other than G and T (i.e., iodo-G or X).
  • RNA oligonucleotide ligand designed to selectively intervene into the process of gene expression will show pharmacological effects in an intact cell .
  • these cell uptake concepts have been used to explain the effectiveness of RNA oligonucleotides as drugs which enhance the effect of interferon treatment (Eppstein D.A., Schryver B.B. & Marsh Y.V. (1986) J. Biol.Chem. 261, 5999) and of the ability of "antisense” or "anti-splice junction" oligonucleotides to selectively inhibit mRNA processing in the cell (Heikkile R. et. al.
  • the design process described herein can be used to design a synthetic DNA oligonucleotide which will bind specifically to any double strand DNA target of interest.
  • the resulting oligonucleotide-duplex DNA complex is best described as a colinear triplex. In the triplex the oligonucleotide molecule occupies the major groove of the duplex. The complex is stabilized by base-base hydrogen bonding at the surface of the major groove, leaving Watson-Crick pairing intact. As a result, the stability and site specificity of the synthetic oligonucleotide is not significantly affected by modification of the phosphodiester linkage or by chemical modification of the oligonucleotide terminus.
  • these oligonucleotides can be chemically modified; enhancing the overall binding stability, increasing the stability with respect to chemical degradation, increasing the rate at which the oligonucleotides are transported into cells, and conferring chemical reactivity to the molecules.
  • Based upon the design method described herein it is possible to design oligonucleotides which are readily taken up by eukaryotic cells and, once in the cell, can be targeted to specific sites within a genome.
  • the site specificity and stability of the synthetic oligonucleotide-target site interaction is as good as current monoclonal antibody-antigen binding interactions.
  • This new class of site specific molecules can be used as gene-specific reagents with the capacity to control the transcription process in a gene-specific fashion. This control is effective on both somatic genes and viral genes which have infected a host cell.
  • synthetic oligonucleotides are appropriately coupled to a reactive chemical complement, it is possible to create a hybrid molecule with the capacity to selectively destroy a gene target of interest.

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Abstract

Selon un procédé de production d'oligonucléotides synthétiques qui se lient à des séquences cibles dans un ADN duplex, formant des triplexes colinéaires par liaison sur la rainure principale, on analyse de l'ADN duplex génomique, on identifie des séquences cibles de nucléotides de plus de 20 nuclétodides environ ayant soit au moins 65% de bases purine environ soit au moins 65% de bases pyrimnidine environ, puis on synthétise les oligonucléotides synthétiques complémentaires des séquences cibles ainsi identifiées. Les oligonucléotides synthétiques ont un G lorsque la position complémentaire de l'ADN duplex présente une paire de bases GC et ont un T lorsque la position complémentaire dans l'ADN duplex présente une paire de bases AT. Les oligonucléotides synthétiques sont orientés de 5' à 3', se lient parallèlement ou en position 3 dans le sens 3' à 5' et se lient anti-parallèlement au brin formé d'au moins 65% de purine. Les oligonucléotides synthétiques ainsi obtenus peuvent être modifiés par modification et/ou remplacement des bases, par adjonction d'agents de liaison et de groupes modifiantsaux terminaux 5' et/ou 3', et par modification de leur structure. Ces oligonucléotides synthétiques se lient à de l'ADN duplex, formant des triplexes. Ce procédé modifie le fonctionnement des gènes ainsi liés et peut être utilisé pour inhiber la croissance cellulaire, pour modifier des taux de protéine, pour traiter des maladies, y compris le cancer, et pour modifier de manière permanente l'ADN.
EP19900901460 1988-12-20 1989-12-20 Triple stranded nucleic acid and methods of use Pending EP0449972A4 (en)

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EP89313391A Expired - Lifetime EP0375408B1 (fr) 1988-12-20 1989-12-20 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

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Families Citing this family (120)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5928863A (en) * 1986-11-26 1999-07-27 Princeton University Triple-stranded nucleic acids
US5422251A (en) * 1986-11-26 1995-06-06 Princeton University Triple-stranded nucleic acids
US5849482A (en) * 1988-09-28 1998-12-15 Epoch Pharmaceuticals, Inc. Crosslinking oligonucleotides
US5721218A (en) * 1989-10-23 1998-02-24 Gilead Sciences, Inc. Oligonucleotides with inverted polarity
US5399676A (en) * 1989-10-23 1995-03-21 Gilead Sciences Oligonucleotides with inverted polarity
IE20010427A1 (en) * 1990-04-10 2003-03-05 Canji Inc Gene therapy for cell proliferative diseases
WO1991018997A1 (fr) * 1990-05-25 1991-12-12 Gilead Sciences, Inc. Agents de reticulation non photoactives, specifiques a une sequence, qui se lient au sillon principal d'adn duplex
EP0463712A3 (en) * 1990-06-27 1992-04-08 University Patents, Inc. Polynucleotide phosphorodithioates as therapeutic agents for retroviral infections
US5734022A (en) * 1990-08-01 1998-03-31 The Johns Hopkins University Antibodies to a novel mammalian protein associated with uncontrolled cell division
WO1992009705A1 (fr) * 1990-11-23 1992-06-11 Gilead Sciences, Inc. Oligomeres formant un triplex et contenant des bases modifiees
WO1992011390A1 (fr) * 1990-12-17 1992-07-09 Idexx Laboratories, Inc. Detection de sequences d'acide nucleique par la formation d'une triple helice de l'adn
US5683874A (en) * 1991-03-27 1997-11-04 Research Corporation Technologies, Inc. Single-stranded circular oligonucleotides capable of forming a triplex with a target sequence
US6136601A (en) * 1991-08-21 2000-10-24 Epoch Pharmaceuticals, Inc. Targeted mutagenesis in living cells using modified oligonucleotides
JPH06510203A (ja) * 1991-08-30 1994-11-17 ザ・ジョーンズ・ホプキンス・ユニバーシティ プリン塩基類似体を含むヌクレオシドオリゴマーを用いる二重鎖ｄｎａの三重らせん複合体の形成
IL103311A0 (en) * 1991-10-07 1993-03-15 Univ Johns Hopkins Formation of triple helix complexes of single stranded nucleic acids using nucleoside oligomers
WO1993009127A1 (fr) * 1991-11-07 1993-05-13 Gilead Sciences, Inc. Formation amelioree d'une triple helice, grace a des oligonucleotides contenant la 2'-desoxy-7-desazaxanthosine, la 2'-desoxy-7-desazaguanosine et des composes analogues
US5594121A (en) * 1991-11-07 1997-01-14 Gilead Sciences, Inc. Enhanced triple-helix and double-helix formation with oligomers containing modified purines
US6235887B1 (en) 1991-11-26 2001-05-22 Isis Pharmaceuticals, Inc. Enhanced triple-helix and double-helix formation directed by oligonucleotides containing modified pyrimidines
TW393513B (en) * 1991-11-26 2000-06-11 Isis Pharmaceuticals Inc Enhanced triple-helix and double-helix formation with oligomers containing modified pyrimidines
AU3222793A (en) * 1991-11-26 1993-06-28 Gilead Sciences, Inc. Enhanced triple-helix and double-helix formation with oligomers containing modified pyrimidines
US5484908A (en) * 1991-11-26 1996-01-16 Gilead Sciences, Inc. Oligonucleotides containing 5-propynyl pyrimidines
US5616461A (en) * 1992-05-14 1997-04-01 Dana-Farber Cancer Institute Assay for antiviral activity using complex of herpesvirus origin of replication and cellular protein
US6403302B1 (en) 1992-09-17 2002-06-11 California Institute Of Technology Methods and compositions for triple helix formation
WO1994007367A1 (fr) * 1992-09-29 1994-04-14 Apollon, Inc. Oligomeres antiviraux liant des voies de polypurine d'arn monocatenaire ou d'hybrides d'arn-adn
EP0674705A4 (fr) * 1992-11-09 1997-11-26 Univ Jefferson Oligonucleotides antisens utilises pour inhiber l'expression de genes de collagene ayant subis une mutation et de type sauvage.
WO1994013793A1 (fr) * 1992-12-04 1994-06-23 Apollon, Inc. Composes et methodes de traitement des leucemies
AU684774B2 (en) * 1992-12-08 1998-01-08 Genta Incorporated Formation of triple helix complexes using a novel motif
WO1994017092A1 (fr) * 1993-01-26 1994-08-04 Microprobe Corporation Oligonucleotides a reticulation bifonctionnelle conçus pour se lier a une sequence de genes souhaitee d'un organisme ou d'une cellule envahisseurs
US6323185B1 (en) 1993-04-23 2001-11-27 The United States Of America As Represented By The Department Of Health And Human Services Anti-viral guanosine-rich oligonucleotides and method of treating HIV
US6288042B1 (en) 1993-04-23 2001-09-11 Aronex Pharmaceuticals, Inc. Anti-viral guanosine-rich tetrad forming oligonucleotides
US5567604A (en) * 1993-04-23 1996-10-22 Aronex Pharmaceuticals, Inc. Anti-viral guanosine-rich oligonucleotides
KR960703170A (ko) 1993-06-23 1996-06-19 알버트 디. 프리센. 안티센스 올리고뉴클레오티드 및 인간면역결핍바이러스감염에서 그것의 치료적이용(antisense oligonucleotides and therapeutic use thereof in human immunodeficiency virus infection)
US5962426A (en) * 1993-06-25 1999-10-05 Yale University Triple-helix forming oligonucleotides for targeted mutagenesis
ATE161541T1 (de) 1993-06-25 1998-01-15 Univ Yale Chemische-modifizierte oligonukleotide für site- spezifisches mutagenesis
WO1995011253A1 (fr) * 1993-10-18 1995-04-27 The Walter And Eliza Hall Institute Of Medical Research Procede destine a amplifier la survie des neurones et agents utiles a cet effet
US5488039A (en) * 1994-01-10 1996-01-30 Abbott Laboratories Method for the production of an enteral formula containing ribo-nucleotides
US5602109A (en) * 1994-01-10 1997-02-11 Abbott Laboratories Method to enhance the immune system of a human
US5492899A (en) * 1994-01-10 1996-02-20 Abbott Laboratories Infant nutritional formula with ribo-nucleotides
US5700590A (en) * 1994-01-10 1997-12-23 Abbott Laboratories Nutritional formula with ribo-nucleotides
US6136858A (en) * 1994-01-10 2000-10-24 Abbott Laboratories Infant formula and methods of improving infant stool patterns
US5808037A (en) * 1995-09-15 1998-09-15 Ramareddy Venkata Guntaka Oligomers which inhibit expression of collagen genes
US6156513A (en) * 1995-09-15 2000-12-05 Ramareddy V. Guntaka Oligmers which inhibit expression of collagen genes
US5780611A (en) * 1995-09-15 1998-07-14 Ramareddy Venkata Guntaka Oligomers which inhibit expression of collagen genes
FR2746412B1 (fr) * 1996-03-21 1998-06-12 Rhone Poulenc Rorer Sa Purification d'adn plasmidique de qualite pharmaceutique
US6312925B1 (en) 1997-05-08 2001-11-06 Epoch Pharmaceuticals, Inc. Methods and compositions to facilitate D-loop formation by oligonucleotides
US6930175B1 (en) 1997-12-12 2005-08-16 The Johns Hopkins University School Of Medicine Gene family with transformation modulating activity
US8114850B2 (en) 1999-04-08 2012-02-14 Advanced Cancer Therapeutics, Llc Antiproliferative activity of G-rich oligonucleotides and method of using same to bind to nucleolin
JP2002541264A (ja) * 1999-04-08 2002-12-03 ユーエイビー・リサーチ・ファウンデーション 冨ｇオリゴヌクレオチドの抗増殖活性およびヌクレオリンに結合するためのその使用方法
US20030114410A1 (en) 2000-08-08 2003-06-19 Technion Research And Development Foundation Ltd. Pharmaceutical compositions and methods useful for modulating angiogenesis and inhibiting metastasis and tumor fibrosis
FR2822156A1 (fr) * 2001-03-16 2002-09-20 Centre Nat Rech Scient Oligonucleotides pour la regulation du gene codant pour le tnf alpha et/ou de genes sous son controle et leurs applications
US8129514B2 (en) 2003-06-19 2012-03-06 Evogene Ltd. Nucleotide sequences for regulating gene expression in plant trichomes and constructs and methods utilizing same
EP1636333A4 (fr) 2003-06-19 2007-10-24 Evogene Ltd Sequences nucleotidiques pour reguler l'expression genetique chez des trichromes vegetaux, et genes hybrides et procedes dans lesquels elles interviennent
ES2435774T3 (es) 2005-07-07 2013-12-23 Yissum Research Development Company, Of The Hebrew University Of Jerusalem Agentes de ácido nucleico para la regulación negativa de H19, y métodos de uso del mismo
US8658608B2 (en) 2005-11-23 2014-02-25 Yale University Modified triple-helix forming oligonucleotides for targeted mutagenesis
WO2008087641A2 (fr) 2007-01-16 2008-07-24 Yissum Research Development Company Of The Hebrew University Of Jerusalem Agents nucléotidiques de silençage de h19 destinés au traitement de l'arthrite rhumatoïde
EP2200628A2 (fr) 2007-07-15 2010-06-30 Yitzchak Hillman Traitement de maladie par des peptides antimicrobien ou leurs inhibiteurs
IL184627A0 (en) 2007-07-15 2008-12-29 Technion Res & Dev Foundation Agents for diagnosing and modulating metastasis and fibrosis as well as inflammation in a mammalian tissue
ES2700141T3 (es) 2007-08-02 2019-02-14 Gilead Biologics Inc Anticuerpos inhibidores LOXL2 y usos de los mismos
US8097712B2 (en) 2007-11-07 2012-01-17 Beelogics Inc. Compositions for conferring tolerance to viral disease in social insects, and the use thereof
WO2009124150A2 (fr) 2008-04-01 2009-10-08 Biosearch Technologies, Inc. Sondes de désactiveur-fluorophore sombres d’acide nucléique stabilisé
AU2009298802A1 (en) 2008-09-23 2010-04-08 Alnylam Pharmaceuticals, Inc. Chemical modifications of monomers and oligonucleotides with cycloaddition
WO2010080769A2 (fr) 2009-01-06 2010-07-15 Arresto Biosciences, Inc. Procédés et compositions chimiothérapeutiques
DK2414542T3 (da) 2009-03-30 2017-11-27 Tel Hashomer Medical Res Infrastructure & Services Ltd Fremgangsmåder til forudsigelse af klinisk forløb for og behandling af multipel sklerose
SG178845A1 (en) 2009-08-21 2012-04-27 Gilead Biologics Inc Catalytic domains from lysyl oxidase and loxl2
MX2012002189A (es) 2009-08-21 2012-03-16 Beeologics Inc Prevencion y cura de las enfermedades de los insectos beneficiosos a traves de moleculas.
WO2011030332A2 (fr) 2009-09-08 2011-03-17 Yeda Research And Development Co. Ltd. Procédés pour la mobilisation de précurseur hématopoïétique
WO2011030329A1 (fr) 2009-09-10 2011-03-17 Yissum Research Development Company Of The Hebrew University Of Jerusalem Ltd. Méthode de traitement de tumeurs
EP3272869B1 (fr) 2009-10-14 2020-06-24 Yissum Research Development Company of The Hebrew University of Jerusalem Ltd. Compositions pour commander les mites varroas dans les abeilles
US8962584B2 (en) 2009-10-14 2015-02-24 Yissum Research Development Company Of The Hebrew University Of Jerusalem, Ltd. Compositions for controlling Varroa mites in bees
BR112012019693A2 (pt) 2010-02-04 2017-06-20 Gilead Biologics Inc anticorpos que se ligam à lisil oxidase-like 2 (loxl2) e métodos de uso para eles.
RU2012148398A (ru) 2010-04-18 2014-05-27 Иеда Рисеч Энд Девелопмент Ко. Лтд. МОЛЕКУЛЫ И СПОСОБЫ ПРИМЕНЕНИЯ МОЛЕКУЛ ДЛЯ ЛЕЧЕНИЯ БОЛЕЗНЕЙ, АССОЦИИРОВАННЫХ С ErbB/ErbB ЛИГАНДАМИ
WO2011133868A2 (fr) 2010-04-22 2011-10-27 Alnylam Pharmaceuticals, Inc. Monomères de dinucléotide et oligonucléotides à conformation restreinte
US10913767B2 (en) 2010-04-22 2021-02-09 Alnylam Pharmaceuticals, Inc. Oligonucleotides comprising acyclic and abasic nucleosides and analogs
WO2011158243A2 (fr) 2010-06-16 2011-12-22 Yissum Research Development Company Of The Hebrew University Of Jerusalem Ltd. Procédé de diagnostic et de traitement du cancer
CN103210082A (zh) 2010-09-15 2013-07-17 雷蒙特亚特特拉维夫大学有限公司 扩增胰岛β细胞和使其再分化的方法
EP2627766B1 (fr) 2010-10-17 2016-05-25 Yeda Research and Development Co. Ltd. Procédés et compositions utilisables en vue du traitement de pathologies médicales associées à l'insuline
US9415103B2 (en) 2011-06-14 2016-08-16 Yeda Research And Development Co. Ltd. Combination therapy to prevent DCIS formation and progression to breast cancer
EP2741753A2 (fr) 2011-08-09 2014-06-18 Yeda Research and Development Co. Ltd. Régulation à la baisse de mir-7 pour favoriser la différenciation des cellules bêta et la production d'insuline
WO2013061328A2 (fr) 2011-10-27 2013-05-02 Yeda Research And Development Co. Ltd. Procédé de traitement du cancer
US9617331B2 (en) 2011-11-27 2017-04-11 Yeda Research And Development Co. Ltd. Methods of regulating angiogenesis by administering agents which increase apoB-100 polypeptide
EP2844745A2 (fr) 2012-02-22 2015-03-11 Brainstem Biotec Ltd. Génération de cellules souches neuronales et de motoneurones
RU2015149680A (ru) 2013-04-21 2017-05-24 Йеда Ресеарч Энд Девелопмент Ко. Лтд. Агенты для подавления активности и/или снижения количества bcl-xl и/или bcl-w
RU2703498C2 (ru) 2013-07-19 2019-10-17 Монсанто Текнолоджи Ллс Композиции и способы борьбы с leptinotarsa
HUE070313T2 (hu) 2013-11-04 2025-05-28 Greenlight Biosciences Inc Ízeltlábú paraziták és kártevõfertõzöttség szabályozására szolgáló készítmények és eljárások
AU2015212341A1 (en) 2014-02-03 2016-09-08 Yissum Research Development Company Of The Hebrew University Of Jerusalem Ltd. Use of Casein kinase I inhibitors for depleting stem cells
BR112016022711A2 (pt) 2014-04-01 2017-10-31 Monsanto Technology Llc composições e métodos para controle de pragas de inseto
RU2021123470A (ru) 2014-07-29 2021-09-06 Монсанто Текнолоджи Ллс Композиции и способы борьбы с насекомыми-вредителями
CN108064274A (zh) 2014-07-30 2018-05-22 耶达研究及发展有限公司 用于培养多能干细胞的培养基
WO2016030899A1 (fr) 2014-08-28 2016-03-03 Yeda Research And Development Co. Ltd. Méthodes de traitement de la sclérose latérale amyotrophique
WO2016079736A2 (fr) 2014-11-17 2016-05-26 Yeda Research And Development Co. Ltd. Procédés de traitement de maladies associées à une fonction mitochondriale
JP6942632B2 (ja) 2015-01-22 2021-09-29 モンサント テクノロジー エルエルシー Ｌｅｐｔｉｎｏｔａｒｓａ防除用組成物及びその方法
EP3261582B1 (fr) 2015-02-26 2021-01-06 Remodeless CV Ltd. Procédés et compositions relatifs à des antagonistes de la leptine
WO2016135732A1 (fr) 2015-02-26 2016-09-01 Yeda Research And Development Co. Ltd. Procédé favorisant la pousse des cheveux
CN107683089B (zh) 2015-05-04 2021-03-16 孟山都技术公司 用于控制节肢动物寄生虫和害虫侵扰的组合物和方法
EP3294280A1 (fr) 2015-05-11 2018-03-21 Yeda Research and Development Co., Ltd. Inhibiteurs de citrine pour le traitement du cancer
WO2016185457A1 (fr) 2015-05-19 2016-11-24 Yeda Research And Development Co. Ltd. Méthodes pour favoriser la lymphagiogenèse
EA201792547A1 (ru) 2015-05-21 2018-04-30 Иеда Рисеч Энд Девелопмент Ко. Лтд. Популяции бактерий для стимуляции состояния здоровья
CA3205381A1 (fr) 2015-07-17 2017-01-26 Alnylam Pharmaceuticals, Inc. Conjugues constitues d'une entite unique a cibles multiples
ES2965461T3 (es) 2015-08-03 2024-04-15 Biokine Therapeutics Ltd Inhibidor de CXCR4 para el tratamiento del cáncer
US11174484B2 (en) 2015-11-10 2021-11-16 B. G. Negev Technologies And Applications Ltd., At Ben- Gurion University Means and methods for reducing tumorigenicity of cancer stem cells
US10828318B2 (en) 2016-01-06 2020-11-10 Yeda Research And Development Co. Ltd. Compositions and methods for treating malignant, autoimmune and inflammatory diseases
JP2019508037A (ja) 2016-02-16 2019-03-28 イェール ユニバーシティーＹａｌｅ Ｕｎｉｖｅｒｓｉｔ 標的化遺伝子編集を増強するための組成物およびその使用方法
US20190216891A1 (en) 2016-03-06 2019-07-18 Yeda Research And Development Co., Ltd. Method for modulating myelination
IL247368A0 (en) 2016-08-18 2016-11-30 Yeda Res & Dev Diagnostic and therapeutic uses of exosomes
WO2018187493A1 (fr) 2017-04-04 2018-10-11 Yale University Compositions et procédés d'administration in utero
EP3658158A4 (fr) 2017-07-27 2021-04-14 The National Institute for Biotechnology in the Negev Ltd. Inhibiteurs de smac/diablo utiles dans le traitement du cancer
IL255664A0 (en) 2017-11-14 2017-12-31 Shachar Idit Hematopoietic stem cells with enhanced properties
EP3723785A1 (fr) 2017-12-17 2020-10-21 Yeda Research and Development Co. Ltd Modulateurs du complexe signalosome cop9 (csn) et leurs utilisations
WO2019155465A1 (fr) 2018-02-08 2019-08-15 Yeda Research And Development Co. Ltd. Procédés d'identification et d'utilisation d'agents pour traiter des maladies associées à un dysfonctionnement de la barrière intestinale
US11648260B2 (en) 2018-03-29 2023-05-16 Technion Research And Development Foundation Limitted Vesicles comprising a PTEN inhibitor and uses of same
MX2020011570A (es) 2018-05-07 2020-11-24 Alnylam Pharmaceuticals Inc Administracion extrahepatica.
WO2020170240A1 (fr) 2019-02-21 2020-08-27 Yissum Research Development Company Of The Hebrew University Of Jerusalem Ltd. Procédé de réduction d'une néphrotoxicité induite par des médicaments
AU2020279101B2 (en) 2019-05-17 2025-07-24 Alnylam Pharmaceuticals, Inc. Oral delivery of oligonucleotides
EP4004548B1 (fr) 2019-07-29 2026-01-14 Yeda Research and Development Co. Ltd Procédés de traitement et de diagnostic du cancer du poumon
WO2022011214A1 (fr) 2020-07-10 2022-01-13 Alnylam Pharmaceuticals, Inc. Parni circulaires
WO2023283403A2 (fr) 2021-07-09 2023-01-12 Alnylam Pharmaceuticals, Inc. Composés bis-arni pour administration au snc
AU2022325816A1 (en) 2021-08-10 2024-03-21 Gamida-Cell Ltd. Engineered nk cells, methods of their production and uses thereof
EP4522742A2 (fr) 2022-05-13 2025-03-19 Alnylam Pharmaceuticals, Inc. Oligonucléotides à boucle simple brin
WO2024137756A1 (fr) * 2022-12-23 2024-06-27 Hongene Biotech Corporation Composés et procédés de synthèse d'oligonucléotides en phase liquide
EP4709855A2 (fr) 2023-05-12 2026-03-18 Alnylam Pharmaceuticals, Inc. Oligonucléotides à boucle simple brin

Non-Patent Citations (8)

* Cited by examiner, † Cited by third party
Title
NATURE, vol. 339, no. 6226, June 22, 1989, New York, P. RAJAGOPAL et al. "Triple-strand formation in the homopurine:homopyrimidine DNA oligonucleotides d(G-A)4 and d(T-C)4", pages 637-640 *
NUCLEIC ACIDS RESEARCH, vol. 14, no. 23, December 9, 1986, Oxford, R.D. DRINKWATER et al. "Two human repetitive DNA elements: a new interspersed repeat found in the factor IX gene, and a satellite 11 tandem repeat sequence", page 9541 *
NUCLEIC ACIDS RESEARCH, vol. 16, no. 24, December 23, 1988, Oxford, J.-C. FRANCOISE et al. "Sequence-specific recog- nition of the major groove of DNA by oligodeoxynucleotides via triple helix formation. Footprinting studies", pages 11431-11440 *
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, vol. 84, no. 15, August 1987, Baltimore, USA, S.L. BROITMAN et al. "Formation of the triple-stranded polynucleotide helix, poly(A A U)", pages 5120-5124 *
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, vol. 85, no. 5, March 1988, Baltimore, USA, D. PRASEUTH et al. "Sequence- -specific binding and photo- crosslinking of alpha and beta oligodeoxynucleotides to the major groove of DNA via triple-helix formation", pages 1349-1353 *
SCIENCE, vol. 238, October 30, 1987, Washington DC, H.E. MOSER et al. "Sequence- Specific Cleavage of Double Helical DNA by Triple Helix Formation", pages 645-650 *
SCIENCE, vol. 241, June 22, 1988, Washington DC, M. COONEY et al. "Site-Specific Oligonucleotide Binding Represses Transcription of the Human c-myc Gene in Vitro", pages 456-459 *
See also references of WO9006934A1 *

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EP0375408B1 (fr) 1995-02-22
JPH04502407A (ja) 1992-05-07
EP0375408A1 (fr) 1990-06-27
PT92640A (pt) 1990-06-29
WO1990006934A1 (fr) 1990-06-28
EP0449972A4 (en) 1992-03-18
ATE118818T1 (de) 1995-03-15
DE68921315D1 (de) 1995-03-30
DE68921315T2 (de) 1995-08-03
AU4838490A (en) 1990-07-10
DK120091D0 (da) 1991-06-20
CA2006008C (fr) 2000-02-15
ES2069598T3 (es) 1995-05-16
AU640910B2 (en) 1993-09-09
DK120091A (da) 1991-06-20
CA2006008A1 (fr) 1990-06-20
PT92640B (pt) 1995-09-12

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