[go: up one dir, main page]
More Web Proxy on the site http://driver.im/

WO2000031302A1 - Synthese binaire in situ de molecules biologiquement efficaces - Google Patents

Synthese binaire in situ de molecules biologiquement efficaces Download PDF

Info

Publication number
WO2000031302A1
WO2000031302A1 PCT/US1999/027681 US9927681W WO0031302A1 WO 2000031302 A1 WO2000031302 A1 WO 2000031302A1 US 9927681 W US9927681 W US 9927681W WO 0031302 A1 WO0031302 A1 WO 0031302A1
Authority
WO
WIPO (PCT)
Prior art keywords
nucleic acid
oligonucleotides
toxin
proximity
oligonucleotide
Prior art date
Application number
PCT/US1999/027681
Other languages
English (en)
Inventor
David J. Ecker
Original Assignee
Isis Pharmaceuticals, Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from US09/200,107 external-priority patent/US6492111B1/en
Application filed by Isis Pharmaceuticals, Inc. filed Critical Isis Pharmaceuticals, Inc.
Priority to AU18272/00A priority Critical patent/AU1827200A/en
Publication of WO2000031302A1 publication Critical patent/WO2000031302A1/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6841—In situ hybridisation
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/35—Nature of the modification
    • C12N2310/351—Conjugate
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention is directed to the synthesis, generation or release of molecules within cells and tissues, which molecules have significant, biological effects. Such molecules are synthesized which permit the identification of the cells or tissues, which destroy the cells or tissues, which moderate, modulate, or enhance cellular or tissue function, or which otherwise have a diagnostic, therapeutic, nutritional or other biological effect.
  • the in situ generation of such molecules is specific to particular cells and tissues such that the molecules thus formed or released are delivered specifically to the cells or tissues in question.
  • Such high specificity of delivery overcomes many therapeutic impediments and can provide high "leverage" with concomitant low side effects.
  • nucleic acid structures present in cells to serve as "targets" for diagnostic and therapeutic regimes.
  • a classical example of such employment is evidenced by the field of antisense therapeutics.
  • a nucleic acid sequence or structure is identified as being associated with the production of a gene product - usually a peptide - which has deleterious effects upon cells or tissues.
  • the target nucleic acid may be an oncogene, an mRNA associated with the genesis or development of a disease state or a hype ⁇ roliferative gene structures which proliferation is to be reduced or eliminated.
  • oligonucleotides are designed which are specifically bindable to a nucleic acids sequence or structure, usually rnRNA, the down regulation of which is desired.
  • the oligonucleotide usually in the form of a chemically modified analog or construct, is administered to the cells containing the targeted nucleic acid.
  • the specific binding of the oligonucleotide interferes with expression of the rnRNA, thus interfering with its function and expression.
  • Protein normally expressed by the rnRNA is either not expressed at all or is expressed in much lower quantity with a concomitant, beneficial therapeutic effect.
  • the paradigm may also be used for diagnosis and research in ways which are well known to persons of ordinary skill in the art.
  • Nucleic acids which can be used to target in accordance with some embodiments of this invention include mRNA molecules, which preferably have secondary structures such as stem-loop structures, or unique secondary structural sites, such as the molecular interaction sites taught in U.S. application Serial Numbers 09/076,440, 09/076,447, and 09/076,404, each of which is inco ⁇ orated herein by reference in its entirety.
  • Potential target nucleic acids include, but are not limited to, mammalian, bacterial, fungal and viral mRNA. Oligonucleotides have been shown to bind to numerous targets including, for example, Candida (U.S. Patent No. 5,691,461), protein kinase C (U.S. Patent Numbers 5,703,054, 5,681,944, and 5,620,963), papillomavirus (U.S. Patent Numbers 5,681,944 and 5,756,282), he ⁇ esvirus (U.S. Patent No. 5,658,891), cytomegalovirus (U.S. Patent Numbers 5,607,923 and 5,595,978), human immunodeficiency virus (U.S. Patent No.
  • Viassov discloses that, along with one oligonucleotide there is a fiourescent sensitizer, while the other oligonucleotide carries a structure which, when irradiated with ultraviolet radiation, is known to react with nucleic acids.
  • Viassov demonstrates a rapid modification of the DNA to which the oligonucleotides were targeted when the two oligonucleotides are allowed to bind to adjacent sites on the DNA and be subsequently irradiated. The proximity of the photosensitizer to the group which transfers energy to the DNA was viewed to be important to the reaction with the DNA.
  • FRET fluorescence resonance energy transfer
  • Oligonucelotides Conjugated to Diimidazole Construction Antisense Nucleic Drug Development, 7(1): 39-42, (1997), prepares oligonucleotides conjugated to a chemical construction having two histidine residues. Yurchenko et al, "Cleavage ofLeishmania Mini - Exon Sequence by Oligonucleotides Conjugated to a Diimidazole Construction ", Nucleosides and Nucleosides, 16 (7-9): 1721-1725, (1996), is directed to similar subject matter.
  • a nucleic acid target which has been identified as being present in the cell or tissue of interest is caused to be the obj ect of specific binding by a plurality of oligonucleotides.
  • a molecular species is either synthesized, formed or released. The molecular species has great biological effect within the cell or tissue which, of course, carries the target nucleic acid.
  • cells having the target nucleic acid may either be identified, imaged, killed, benefitted or modified in some significant way.
  • this paradigm is denominated "in situ binary synthesis,” since a molecule having biological activity is created upon the specific binding of oligonucleotides to the target nucleic acid.
  • the method is in situ since the formation, synthesis or release of the molecule takes place in situ - within the cell or tissue.
  • the methodologies of the present invention do not necessarily involve modification of nucleic acid, or at least not the nucleic acid which serves as a targeting nucleic acid.
  • the objective of a specific binding of an oligonucleotide is not necessarily destruction or inhibition of the nucleic acid, usually mRNA, which has been targeted. Rather, the nucleic acid simply serves as the "locator beacon" and as a place for the docking of oligonucleotides by virtue of their specific binding.
  • the ability of a plurality of oligonucleotides to specifically bind to a target nucleic acid at adjacent sites upon the nucleic acid permits the generation, synthesis or release of the biologically effective molecules in a very highly controlled fashion.
  • generation, synthesis or release of the biological molecule does not take place.
  • This high degree of control contributes to the utility of the present invention in that extraordinary toxic or potent molecules may be used without the worry of accidental generation or release of such molecules at locations other than the ones desired.
  • the present invention builds upon previous methodologies of oligonucleotide interaction with cellular nucleic acids, especially mRNA in the following way. It is well known to identify nucleic acids structures or sequences which are related to disease states. It is also known how to prepare oligonucleotides which have antisense sequences to the nucleic acids whose function or activity is to be moderated or ended. Moreover, a wide variety of chemical modifications of nucleic acids, and, indeed, a host of oligonucleotide analogs are known for this pu ⁇ ose.
  • nucleic acid it is essential that the target nucleic acid be one which is important to disease states. It is not enough, under these prime paradigms, for the nucleic acid merely to be associated with a cell or tissue which is in a disease state. It may be immediately seen that interaction with a non-critical nucleic acid, such as an mRNA, even if very specific and entirely efficacious, will not lead to any beneficial results since the expression of the mRNA is not vital to survival of the cell or tissue. Moreover, antisense interaction with nucleic acids, while much more efficient than small molecule interaction with peptides, still requires the individual, specific binding of oligonucleotide to individual nucleic acids, such as mRNAs.
  • the present invention does not require the identification of a target nucleic acid which is vital to the function of a cell on which is causative of a disease state. Indeed the nucleic acid thus identified need not actually be translated into protein in large quantities within the cell or tissue in question. Rather, it is only necessary that the target nucleic acid be associated with or predictive of the existence of the disease state or other state of interest in cells or tissues to be treated. This is so because it is not the inactivation of the nucleic which is sought, but rather the use of that nucleic acid to target oligonucleotides to the cells or tissues which contain the target nucleic acid.
  • a plurality of oligonucleotides are formulated to be specifically bindable to the target nucleic acid. Moreover, such oligonucleotides are formulated to have sequences which cause them to specifically bind to the target nucleic acid at adjacent sites. While it is possible that the oligonucleotides could bind at sites which are removed by one or two bases, it is greatly preferred that the oligonucleotides bind immediately adjacent to each other. It will, thus, be seen that the present methodology causes two oligonucleotides to enter a cell, tissue, or preparation having target nucleic acid and to bind to the target nucleic acid "right next to” each other. This close, predicable, and regulatable proximity of oligonucleotides to each other that contributes to the present invention.
  • a first one of the oligonucleotides carries with it a first portion of the biologically effective molecule to be synthesized, formulated or released.
  • the second oligonucleotide carries with it a second portion of the biologically effective molecule. It is only when the first and second portions of the molecules are brought together in physical proximity and, preferably, with careful geometric alignment, that the biological molecule is either synthesized, generated, or released.
  • the biologically effective molecule is actually synthesized through the joining together with covalent bonds of two or more fragments or synthons thereof, whether a precursor molecule is transformed, modified, or cleaved during this process, or whether a pre-formed, biologically effective molecule is cleaved or released from the oligonucleotides through the present methodology, the biologically effected molecule is not rendered effective unless and until the two oligonucleotides specifically bind at adjacent sites on the target nucleic acid within the cell, tissue, or in vitro preparation.
  • first oligonucleotide carries with it a first synthon while the second oligonucleotide carries with or on it, or otherwise comprises, a second synthon.
  • first synthon is extraordinarily broad and means a chemical moiety which is capable of interacting with another chemical moiety to give rise to chemical alteration.
  • an oligonucleotide may specifically bind to the target nucleic acid followed by the interaction of the heteroduplex with another species, which is not an oligonucleotide.
  • Such other species may be, for example, a peptide, a carbohydrate or complex sugar, or a "small" molecule, e.g. a non-oligomer.
  • Certain aspects of the invention provide for the employment of two or more non-oligonucleotides in the practice hereof.
  • specific binding of two or more non- oligonucleotide oligomers, carbohydrates or sugars, or small molecules may serve the pu ⁇ oses described herein.
  • two or more small molecules could bind specifically and adj acently to the target nucleic acid, giving rise to a biologically active molecular species for, e.g. destruction of the cell containing the target nucleic acid or detection thereof.
  • the present invention provides for the detection of cells or nucleic acids found in the cells, with the killing of cells having target nucleic acids, and with certain other beneficial results. While, in all such cases, it may be seen that the respective oligonucleotides carry with them synthons, in accordance with the broad definition of that term, it may also be convenient to refer to the chemical moieties carried by the respective oligonucleotides in terms which are more nearly suited to their function.
  • the respective synthons carried by the oligonucleotides may be referred to as "identiphores.”
  • the identiphores are moieties which can interact with each other to give rise to either an interaction or a molecular species which can be detected in some way.
  • Detection may take place using any form of spectroscopy, x-ray examination, biochemical or biological analyses or otherwise. What is required is that the confluence of the two identiphores carried, respectively, by the oligonucleotides which specifically bind to the target nucleic acid, be detectable. The presence of the detectable moiety formed, synthesized or released by the conjunction in space of the two identiphores is, according to this embodiment, probative of the presence of the target nucleic acid in the cell, tissue or in vitro assay.
  • x-ray detection may be employed. This is particularly beneficial when the identiphore contains a heavy metal such as a heavy metal contained within an heterocyclic structure such as apo ⁇ hyrin, heme or similar species.
  • the identiphores may form a charged transfer complex when in proximity with each other which complex may be detectable through spectroscopy. Such identiphores may also become cobandedly bonded with each other upon coming into proximity giving rise to a chemical moiety or molecule which can, itself be detected.
  • Other forms of detection including magnetic resonance spectroscopy, proton magnetic resonance spectroscopy, electron resonance spectroscopy, fluorescence spectroscopy or electromagnetic spectroscopy may also be profitable employed in connection with this embodiment of the invention.
  • the identiphores can comprise portions of a charge transfer complex of TTF-TCNQ, (Tetrathiafulvalene and Tetracyanoquinodimethane).
  • TTF-TCNQ Tetrathiafulvalene and Tetracyanoquinodimethane
  • Bis-(ethylenedithio)dithiapyrene (ETDTYP) and Dibenzo barreleno tetracyano quino dimethane or Triphenylphosphine and Acrylonitrile could form such complexes.
  • Other charge transfer complexes are known to persons of ordinary skill in the art.
  • these molecular moieties may be caused to comprise the oligonucleotides which specifically bind to the target nucleic acid. Bringing the two identiphores into proximity gives rise to the detectable complex.
  • identiphores for identifying the presence of target nucleic acid in cells or tissues may be used to diagnose certain cellular biological states such as disease states, especially hype ⁇ roliferative states and cancers.
  • the identiphores are best placed into spacial proximity with each other and also to have a preselected geometric relationship one to the other. This is especially true than intermolecular interactions are required such as charge transfer complexes and the like.
  • This aspect of the invention provides methods for identifying cells containing a preselected nucleic acid sequence. These methods comprise contacting the cells with a first oligonucleotide specifically bindable with the preselected nucleic acid, the first oligonucleotide comprising a first identiphore. The cells are also contacted with the second oligonucleotide specifically bindable with the preselected nucleic acid, preferably at a site immediately adjacent to the site where the first oligonucleotide has bound to the nucleic acid. The second oligonucleotide also comprises an identiphore. The first and second identiphores are detectable when in proximity with each other.
  • oligonucleotides may either react with each other to form a new molecular species, cause a species to be released from the oligonucleotides for detection, or may give rise to an entirely new moiety which, itself, may be detected in any of the ways known to persons of ordinary skill in the art.
  • the synthons carried by the respective oligonucleotides may give rise to toxic molecules.
  • Such molecules may either be synthesized, generated or released upon the specific binding of the two oligonucleotides to adjacent sites on the target nucleic acid.
  • the generation of a toxic molecule as a result of this specific binding gives rise to cell death and possibly even destruction of tissue material in adjacent cells.
  • many highly toxic molecules exist in nature and otherwise which can effectuate widespread destruction on a cellular and tissue level. Prior therapeutic regimes have never been able to exploit this phenomenon, since systemic or untargeted toxicity has never been able to be avoided heretofore.
  • the present invention permits this.
  • the present invention provides methods of killing cells, which cells contain a preselected nucleic acid.
  • the method comprises contacting the cells with a first oligonucleotide specifically bindable with the selected preselected nucleic acid.
  • the first oligonucleotide comprises a first toxiphore.
  • toxiphore is meant herein to mean a chemical species which, when combined with one or more additional toxiphores, gives rise to a toxic molecular species, interaction or phenomenon. Such toxicity is to be expressed on a cellular or tissue level; it is not necessary that any particular nucleic acid be inactivated or "killed” thereby.
  • the method further comprises contacting these preselected nucleic acid sequence with a second oligonucleotide specifically bindable with it.
  • the second oligonucleotide contains a second toxiphore.
  • the first and second toxiphores either synthesize, generate or release a toxin upon coming into proximity with each other as is the case when the two oligonucleotides, carrying their toxiphores, specifically bind to adjacent sites on the target nucleic acid.
  • the toxiphores be placed into spatial proximity when the oligonucleotides specifically bind to the target nucleic acids. Additionally, it is greatly preferred that the toxiphores be oriented geometrically so as to facilitate the formation of the molecular species, its generation or release. In accordance with preferred embodiments, the toxiphores become chemically bonded one to the other to give rise to the toxic, molecular specie. Such specie is, of course, highly biologically effective. It is also within the spirit of this invention that one toxiphore modify the second toxiphore to give rise to the biologically effective, toxic molecule.
  • One toxiphore may also cleave from the oligonucleotide, the heteroduplex, or otherwise, a toxic species.
  • the preselected nucleic acid, to which the oligonucleotides and their related toxiphores will specifically attach be predictive of a cellular biological state.
  • Such state is usually a disease state, especially a hype ⁇ roliferative state or one indicative of cancer.
  • Other cellular or tissues states may also be associated with the preselected nucleic acid and, indeed, the present invention may be used in the sense of a probe as well as in the sense of a therapeutic or diagnostic. A host of research functions may also be benefitted through use of the present invention.
  • the toxic molecule which, in accordance with preferred embodiments of this invention, are synthesized, generated or released may be anything which has effective, preferably powerful cellular toxicity within related tissues or organs.
  • the toxin may be a peptide such as ricin, sarcin, or diphtheria, botulism, etc. toxins. In such cases, the toxin would comprise a functional domain of the toxin.
  • the toxin may be bacterial.
  • the toxin need not be a peptide.
  • the toxin could be an amphibian toxin such as tetradotoxin or bufotoxin.
  • the toxins may also be from invertebrates, such as arachnids, or from reptiles or amphibians. In short, any number of toxins may be employed in conjunction with the present invention. It is preferred that the toxin be very powerful such that only a few, perhaps as few as one or two molecules of the toxin are necessary to effect cell death. Numbers of toxin molecules fewer than ten are preferred in accordance with certain embodiments to kill any given cell. As will be appreciated, a preferred embodiment of the present aspect of the invention is for the treatment of disease state in mammals. Thus, a tissue of a mammal is contacted with the oligonucleotides having toxiphores associated therewith under conditions such that heteroduplexes can be formed.
  • heteroduplex formation is encouraged through appropriate selection of reaction conditions.
  • the toxiphores Upon the specific binding of the oligonucleotides to the preselected nucleic acid, the toxiphores interact with each other to give rise to a toxic molecule or other toxic effect.
  • the toxin is a peptide
  • the activation energy required for the formation of the peptide bond may be significantly lowered such that spontaneous generation of the peptide bond occurs.
  • the resulting, toxin is then either active immediately or can be released or cleaved from the complex for toxic activity.
  • Organic molecules other than peptides may also be prepared in this way, cleaved, modified, or released.
  • the chemical species which effects the toxic activity may also be other than an organic species; they may be organometallic or even inorganic.
  • one toxiphore might contain a complex metal ion which, in uncomplexed form or in a different oxidation state is highly toxic.
  • the interaction with the second toxiphore may give rise to the toxic form of the metal ion or to its complex.
  • a number of "heavy" metallic species and some which are not so “heavy” are known to be highly toxic in one or another form. All of these are contemplated by the present invention.
  • the present invention provides for the in situ synthesis of chemical species. This is accomplished through specifically binding first and second oligonucleotides to a preselected nucleic acid at adjacent sites of the nucleic acid. First and second sythons, related to or forming part of the respective oligonucleotides, are then reacted with each other or caused to interact to give rise to a new molecular species or an altered form of an existing molecular species. Significant biological effects may thus be generated upon the cell or tissues comprising those cells.
  • peptides may be prepared by bringing together two synthons comprising peptide residues, which residues, when carefully oriented in space and oriented as to geometry may form the peptide bond either spontaneously or through the intervention of a catalyst.
  • a catalytic agent may also be included in or more of the oligonucleotides.
  • a host of other organic molecules may similarly be formed as will be apparent to persons of ordinary skill in the art.
  • Organometallic materials may also be prepared hereby and the oxidation state of organic, and organometallic species may be changed through intermediation of oxidizing and reducing agents forming part or all of one or both of the synthons.
  • the charged transfer complexes as discussed herein before may also be placed into apposition and may be used in either a catalytic, synthetic, biologically active or other functional applications.
  • the ability of the present invention to orient synthons in precise proximity and in exact geometric relationship one with the other permits the overcoming of activation energies which would otherwise apply. Accordingly, even reactions which seem extraordinarily slow under solution conditions, may run at sensible rates when synthesized in accordance with the present invention.
  • the present invention provides for the preparation of such chemical species in amounts which are detectable in cells containing the target nucleic acid. It is, of course, preferred that such nucleic acid be predictive of a biological condition or disease state such that the preparation and delivery of the molecules thus formed or furnished is delivered in situ to such cells. Diagnosis, therapy, or killing of the cell may ensue.
  • the present invention also provides compositions of matter.
  • the invention provides a pair of oligonucleotides each of which is specifically bindable with a preselected nucleic acid.
  • the binding of the pair of oligonucleotides is preferably at adjacent sites on the preselected nucleic acid.
  • Each of the oligonucleotides comprises a portion of a molecular species, which species is formed when the adjacent, specific binding of the oligonucleotides to the nucleic acid occurs. Any of the foregoing methodologies may give rise to pairs of oligonucleotides which are contemplated by the present invention.
  • the present invention may also be applied to additional oligonucleotides with additional synthons (toxiphores, identiphores, etc.) whereupon binding of three or more oligonucleotides to adjacent sites on the target nucleic acid is accomplished with results analogous to those described herein above.
  • the oligonucleotides may give rise to toxins, charge transfer complexes, detectable molecules for detection and sensing, cell regulatory moieties, nutritional species, and to a wide variety of other compounds having biological activity.
  • the present invention is also directed to compositions of matter comprising a pair of oligonucleotides as herein above together with a diluent or carrier, especially a pharmaceutically acceptable diluent or carrier.
  • oligonucleotide has been used very broadly. Persons of ordinary skill in art will appreciate that the term includes wild type oligonucleotides, those which are unmodified in any way in terms of their chemical construction or substituents. The term also includes semisynthetic and modified oligonucleotides and molecules which are analogous to oligonucleotides.
  • oligonucleotide includes peptide nucleic acids, "PNAs", molecules which are analogous in function and spatial relationships to nucleic acids and which may be used in oligonucleotide therapeutics such as antisense and the like. These have been well-characterized in the literature and are set forth in number of U.S. patents, many of which are assigned to the assignee of the present application. Each of these are inco ⁇ orated herein by reference.
  • peptide nucleic acids U.S. Patent Numbers 5,641,625, 5,700,922, 5,719,262, 5,714,331, 5,766,855, 5,773,571, 5,786,461, and 5,736,336, each of which is inco ⁇ orated herein by reference in its entirety
  • ribozymes U.S. Patent Numbers 5,599,706, 5,801,158, 5,639,655, 5,635,385, 5,599,704, 5,610,052, 5,766,942, and 5,747,335, each of which is inco ⁇ orated herein by reference in its entirety
  • small molecules U.S.
  • Chemically modified oligonucleotides include those which are modified in the backbone, e.g. phosphorothioate, methylphosphonates and a host of other backbone modifications as well as modifications to the substituents on the sugar rings present in the oligomers. Further modifications may be had on the base structure in a number of ways. These are exemplified by a number of U.S. patents including many owned by the assignee of the present application.
  • oligonucleotide carrying a synthon (identiphore, toxiphore, etc.) may be caused to bind specifically to a site of a target nucleic acid.
  • a synthon identityphore, toxiphore, etc.
  • Another biooligomer may then be caused to bind to the heteroduplex thus formed. This latter oligomer need not necessarily be an oligonucleotide.
  • certain peptides are known to be able to complex with heteroduplexes formed between a nucleic acids and oligonucleotides. It is possible to include a synthon along with such peptide and to cause the same to bind the heteroduplex thus bringing the synthon of the peptide and the synthon carried by the oligonucleotide into proximity with the results explained above. Many other variations are possible in accordance with the full spirit of the present invention.
  • a single oligonucleotide to accomplish the function presently exemplified through a plurality of oligonucleotides.
  • a single oligonucleotide having two portions, each of the two portions carrying a synthon (identiphore, toxiphore, etc.) may be employed. If the two portions of the single oligonucleotide can bind at adjacent locations on the target nucleic acid, thus bringing the synthons into proximity, the spirit of the present invention may be accomplished.
  • oligomer which first binds with the target nucleic acid and then binds with the heterodymer formed by the first part of the oligomer and the target nucleic acid could be used in conjunction with certain embodiments of the present invention.

Landscapes

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

Abstract

On provoque la fixation d'une pluralité d'oligonucléotides sur un acide nucléique cible spécifique qui prédit un état pathologique ou un état biologique dans des cellules contenant cet acide nucléique cible. Chaque oligonucléotide, qui peut être fonctionnalisé ou qui peut être présent sous forme d'analogue d'oligonucléotide, comprend une pluralité de synthons. Ces synthons, qui peuvent être des identiphores, des toxiphores, ou d'autres précurseurs de molécules biologiquement efficaces, interagissent lorsqu'une fixation de chaque oligonucléotide a lieu sur des sites adjacents les uns aux autres sur l'acide nucléique cible. L'interaction qui en résulte provoque la synthèse et la génération ou la libération de molécules à haute activité biologique in situ dans la cellule dans laquelle la fixation spécifique a lieu. Il est ainsi possible d'utiliser les molécules à très haute toxicité pour tuer les cellules contenant les acides nucléiques cibles. L'invention concerne également un procédé d'imagerie et d'autres utilisations.
PCT/US1999/027681 1998-11-25 1999-11-22 Synthese binaire in situ de molecules biologiquement efficaces WO2000031302A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU18272/00A AU1827200A (en) 1998-11-25 1999-11-22 (in situ) binary synthesis of biologically effective molecules

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US11002498P 1998-11-25 1998-11-25
US09/200,107 1998-11-25
US09/200,107 US6492111B1 (en) 1998-11-25 1998-11-25 In situ binary synthesis of biologically effective molecules
US60/110,024 1998-11-25

Publications (1)

Publication Number Publication Date
WO2000031302A1 true WO2000031302A1 (fr) 2000-06-02

Family

ID=26807629

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1999/027681 WO2000031302A1 (fr) 1998-11-25 1999-11-22 Synthese binaire in situ de molecules biologiquement efficaces

Country Status (2)

Country Link
AU (1) AU1827200A (fr)
WO (1) WO2000031302A1 (fr)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1546327A1 (fr) * 1999-11-23 2005-06-29 Chromocell Corporation Selection et isolement de cellules vivantes au moyen de sondes liant l'arn
US8916503B2 (en) 2004-02-18 2014-12-23 Chromocell Corporation Methods and materials using signaling probes

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0246864A2 (fr) * 1986-05-19 1987-11-25 Bio-Rad Laboratories, Inc. Sondes d'hybridation
WO1993017128A1 (fr) * 1992-02-28 1993-09-02 Amoco Corporation Procedes de detection de structure et de reagencements chromosomiques
US5424413A (en) * 1992-01-22 1995-06-13 Gen-Probe Incorporated Branched nucleic acid probes
WO1995019449A1 (fr) * 1994-01-14 1995-07-20 Aprogenex, Inc. Populations de sondes d'hybridation non adjacentes
US5874555A (en) * 1987-10-30 1999-02-23 California Institute Of Technology Triple helices and processes for making same

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0246864A2 (fr) * 1986-05-19 1987-11-25 Bio-Rad Laboratories, Inc. Sondes d'hybridation
US5874555A (en) * 1987-10-30 1999-02-23 California Institute Of Technology Triple helices and processes for making same
US5424413A (en) * 1992-01-22 1995-06-13 Gen-Probe Incorporated Branched nucleic acid probes
WO1993017128A1 (fr) * 1992-02-28 1993-09-02 Amoco Corporation Procedes de detection de structure et de reagencements chromosomiques
WO1995019449A1 (fr) * 1994-01-14 1995-07-20 Aprogenex, Inc. Populations de sondes d'hybridation non adjacentes

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
CHOE ET AL.: "The Crystal Structure of Diptheria Toxin", NATURE, vol. 257, 21 May 1992 (1992-05-21), pages 216 - 222, XP002926823 *
SACCHI N. ET AL.: "Interphase Cytogenetics of the t(8;21)(q22;q22) Associated with Acute Myelogenous Leukemia by Two-color Fluorescence In Situ Hybridization", CANCER GENETICS CYTOGENETICS, vol. 79, 1995, pages 97 - 103, XP002926819 *
SIXOU S. ET AL.: "Intracellular Oligonucleotide Hybridization Detected by Fluorescence Resonance Energy Transfer (FRET)", NUCLEIC ACIDS RESEARCH, vol. 22, no. 4, 1994, pages 662 - 668, XP002926820 *
WAHLSTEN J. L. ET AL.: "Separation of Function Between the Domains of Toxic Shock Syndrome Toxin-1", THE JOURNAL OF IMMUNOLOGY, vol. 160, 1998, pages 854 - 859, XP002926822 *
WILLIAMS M. D. ET AL.: "Production of Recombinant DTctGMCSF Fusion Toxin in a Baculovirus Expression Vector System for Biotherapy of GMCSF-Receptor Positive Hematologic Malignancies", PROTEIN EXPRESSION AND PURIFICATION, vol. 13, 1998, pages 210 - 221, XP002926824 *
WOLF ET AL.: "Regeneration of Active Receptor Recognition Domains on the B Subunit of Cholera Toxin by Formation of Hybrids from Chemically Inactivated Derivatives", BIOCHIMICA ET BIOPHYSIC ACTA, vol. 1223, 1994, pages 285 - 295, XP002926821 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1546327A1 (fr) * 1999-11-23 2005-06-29 Chromocell Corporation Selection et isolement de cellules vivantes au moyen de sondes liant l'arn
EP1546327A4 (fr) * 1999-11-23 2005-11-09 Chromocell Corp Selection et isolement de cellules vivantes au moyen de sondes liant l'arn
US8916503B2 (en) 2004-02-18 2014-12-23 Chromocell Corporation Methods and materials using signaling probes

Also Published As

Publication number Publication date
AU1827200A (en) 2000-06-13

Similar Documents

Publication Publication Date Title
White et al. Picazoplatin, an azide-containing platinum (II) derivative for target analysis by click chemistry
US6713456B1 (en) Nucleozymes
US5594136A (en) Texaphyrin solid supports and devices
US5561043A (en) Self-assembling multimeric nucleic acid constructs
KR100355251B1 (ko) 텍사피린을사용한rna광절단방법
Rothenberg et al. Oligodeoxynucleotides as anti-sense inhibitors of gene expression: therapeutic implications
WO1991014696A1 (fr) Conjugues a base de disulfure d'oligonucleotide et d'un agent de transport
JPH11506452A (ja) 金属錯体を用いた核酸の加水分解および光切断
Musumeci et al. G-quadruplex-forming oligonucleotide conjugated to magnetic nanoparticles: synthesis, characterization, and enzymatic stability assays
WO2000026226A1 (fr) Detecteurs moleculaires polynucleotidiques a domaines multiples
CA2328599A1 (fr) Dispositifs multimoleculaires, systemes d'administration de medicaments et selection de molecule unique
WO1997033622A2 (fr) Apport d'acides nucleiques par des porphyrines
EP0702685A1 (fr) Hydrolyse d'esters realisee par mediation par des complexes metalliques de texaphyrine
Novopashina et al. Recent advances in the synthesis of high boron-loaded nucleic acids for BNCT
Roth et al. Nucleic acid biotechnology
US20060074034A1 (en) Cobalamin mediated delivery of nucleic acids, analogs and derivatives thereof
Summerton Intracellular inactivation of specific nucleotide sequences: a general approach to the treatment of viral diseases and virally-mediated cancers
Nedorezova et al. Deoxyribozyme‐Based DNA Machines for Cancer Therapy
Sanchez et al. Conjugation reactions involving maleimides and phosphorothioate oligonucleotides
Jing et al. Rational drug design of G-quartet DNA as anti-cancer agents
Murchie et al. T4 endonuclease VII cleaves DNA containing a cisplatin adduct
US6492111B1 (en) In situ binary synthesis of biologically effective molecules
WO2000031302A1 (fr) Synthese binaire in situ de molecules biologiquement efficaces
US5763172A (en) Method of phosphate ester hydrolysis
Zhang et al. G-Quadruplex mRNAs Silencing with Inducible Ribonuclease Targeting Chimera for Precision Tumor Therapy

Legal Events

Date Code Title Description
ENP Entry into the national phase

Ref country code: AU

Ref document number: 2000 18272

Kind code of ref document: A

Format of ref document f/p: F

AK Designated states

Kind code of ref document: A1

Designated state(s): AE AL AM AT AU AZ BA BB BG BR BY CA CH CN CR CU CZ DE DK DM EE ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MD MG MK MN MW MX NO NZ PL PT RO RU SD SE SG SI SK SL TJ TM TR TT UA UG US UZ VN YU ZA ZW

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): GH GM KE LS MW SD SL SZ TZ UG ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE BF BJ CF CG CI CM GA GN GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
REG Reference to national code

Ref country code: DE

Ref legal event code: 8642

122 Ep: pct application non-entry in european phase