[go: up one dir, main page]

WO1994011494A1 - Oligonucleotides antisens utilises pour inhiber l'expression de genes de collagene ayant subis une mutation et de type sauvage - Google Patents

Oligonucleotides antisens utilises pour inhiber l'expression de genes de collagene ayant subis une mutation et de type sauvage Download PDF

Info

Publication number
WO1994011494A1
WO1994011494A1 PCT/US1993/010756 US9310756W WO9411494A1 WO 1994011494 A1 WO1994011494 A1 WO 1994011494A1 US 9310756 W US9310756 W US 9310756W WO 9411494 A1 WO9411494 A1 WO 9411494A1
Authority
WO
WIPO (PCT)
Prior art keywords
collagen
seq
oligonucleotide
sequence
type
Prior art date
Application number
PCT/US1993/010756
Other languages
English (en)
Inventor
Darwin Prockop
Alain Colige
Renato Baserga
Paul Nugent
Original Assignee
Thomas Jefferson University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Thomas Jefferson University filed Critical Thomas Jefferson University
Priority to US08/432,158 priority Critical patent/US5861502A/en
Priority to EP94901327A priority patent/EP0674705A4/fr
Priority to JP6512264A priority patent/JPH08503366A/ja
Publication of WO1994011494A1 publication Critical patent/WO1994011494A1/fr

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • 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
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/31—Chemical structure of the backbone
    • C12N2310/315—Phosphorothioates

Definitions

  • Mutations in either of the two genes for type I procollagen cause osteogenesis imperfecta and a subset of osteoporosis; mutations in the gene for type II procollagen (C0L2A1) cause chondrodysplasias and some forms of osteoarthritis; and mutations in the gene for type III procollagen (COL3A1) cause Ehlers-Danlos syndrome type IV and aneurysms.
  • Most of the mutations of procollagen genes produce disease phenotypes by directing synthesis of structurally abnormal but partially functional proot chains of type I, type II or type III procollagen. The partially functional pro ⁇ chains associate with and become disulfide- linked to normal proo; chains.
  • the mutant chains can have one of several major effects.
  • Kuivaniemi H. et al. , FASEB J. 1991, 5, 2052-2060.
  • One effect is to prevent folding of the three chains into a collagen triple helix and thereby cause degradation of both the abnormal and normal pro ⁇ chains through a process referred to as procollagen suicide.
  • the second effect is to produce minor changes in the conformation of the collagen triple helix and thereby generate mutated monomers that interfere with self-assembly of normal monomers synthesized by the same cells.
  • a third effect of mutations in collagen genes is to decrease the amounts of collagen synthesized by fibroblasts and related cells. Mutations that decrease collagen synthesis, however, cause the relatively mild disease known as type I osteogenesis imperfecta.
  • transgenic mice expressing mutated genes for type I procollagen Deleterious effects of mutant collagen gene expression have been demonstrated in transgenic mice expressing mutated genes for type I procollagen. These transgenic mice developed phenotypes resembling human osteogenesis imperfecta, Stacey, A. et al. , Nature 1988, 332, 131-136; Khillan, J. S. et al. , J. Biol . Chem. 1991, 266, 23373-23379. Further, it has been demonstrated that transgenic mice expressing mutated genes of type II procollagen developed phenotypes resembling human chondrodysplasia. Vandenberg et al., Proc . Natl . Acad. Sci . 1991, 88, 7640-7644.
  • liver cirrhosis is a two-step process in which normal liver tissue is first destroyed by a virus or by alcohol and other toxins, and then excessive amounts of collagen fibers replace the damaged cells before normal liver cell regeneration.
  • Idiopathic pulmonary fibrosis is a lethal condition in which, for largely unknown reasons, normal lung tissue is gradually replaced by excessive amounts of collagen fibers.
  • Progressive systemic sclerosis (scleroderma) is a frequently lethal disease where, again for unknown reasons, skin and many internal organs become leather-like because of excessive depositions of collagen fibers.
  • modified antisense oligonucleotides that are complementary to specific RNAs can inhibit the expression of a number of cellular and viral genes as proteins. See Erickson, R. P., and Izant, J. G. Gene Regulation : Biology Of Antisense RNA And DNA, Vol. 1, Raven Press, New York, 1992. For example, selective inhibition of a p21 gene that differed from a normal gene by a single nucleotide has been reported. Chang, E. H. et al. , Biochemistry 1991, 30, 8283-8286. Moreover, mRNA splice junctions were suitable targets for antisense nucleic acids. Kole, R. et al., Adv. Drug Delivery Rev.
  • Mutations in genes encoding procollagen cause osteogenesis imperfecta, chondrodysplasia and related disorders, and Ehlers-Danlos syndrome type IV. They also cause a subset of osteoporosis, a subset of osteoarthritis and a subset of aneurysms.
  • therapeutic and pharmacologic agents for the treatment of genetic diseases of collagen are few, and none involve the selective inhibition of the expression of the mutant collagen gene causing the disease.
  • excess synthesis and deposition of collagen in the form of scars and fibrous tissue causes most of the deleterious effects of diseases such as liver cirrhosis, pulmonary fibrosis, scleroderma, hypertrophic scarring and keloid formation.
  • the present invention provides a means based on antisense strategies to inhibit selectively expression of either a mutated or a normal gene for collagen. Therefore, it provides a means for preventing or reversing many of these conditions.
  • modified antisense oligonucleotides complementary to an exogenous mutated COL1A1 gene were prepared.
  • the exogenous gene consisted of a construct of the human C0L1A1 gene which was transfected into mouse cells.
  • the mouse cells synthesized mutated pro ⁇ l(I) chains of human type I procollagen.
  • the mouse cells also synthesized normal pro ⁇ l(I) chains of mouse type I procollagen from the endogenous mouse C0L1A1 gene.
  • the modified antisense oligonucleotides were designed to contain a base sequence that was complementary to and that, therefore, would bind to a target sequence in RNA transcripts of the exogenous human COL1A1 gene.
  • the target sequence was 20 nucleotides from exon 1 and intron 1 of the normal human C0L1A1 gene that differed by 9 nucleotides in the same sequence of the normal mouse COL1A1 gene.
  • Missense and sense versions of the same oligonucleotide had essentially no effect on expression of the exogenous gene.
  • the inhibition observed with the most effective oligonucleotide was reduced by introducing a single base change in the oligonucleotide.
  • Selective inhibition of expression of the exogenous collagen gene was consistently observed in all experiments.
  • the concentration of oligonucleotide required for effective inhibition was as low as 0.1 ⁇ M.
  • oligonucleotides designed to target sequences in normal collagen genes may be useful in diseases and related conditions in which deleterious effects are produced by excessive synthesis and deposition of collagen in tissues.
  • Oligonucleotides complementary to specific sequences in either the human C0L1A1 gene or the mouse C0L1A1 gene are provided.
  • Methods are also provided for selecting and preparing the oligonucleotides. Further, methods are included in the invention for treating mammals having diseases or related conditions caused by expression of mutated gene for collagen or caused by excessive expression of normal collagen genes in response to injury to specific tissues.
  • the methods of the invention will be particularly useful to treat humans suffering from diseases of collagen by selective inhibition of mutant collagen gene expression using oligonucleotides of the invention. They will also be useful to treat humans and other mammals suffering from diseases and related conditions caused by excessive synthesis and deposition of normal collagen in tissues, i.e., condition such as liver cirrhosis, pulmonary fibrosis, scleroderma and scarring following trauma or surgery.
  • Figure 1 shows Western blot assays of expression of the endogenous gene (SEQ ID NO: 4) and the exogenous gene (SEQ ID NO: 3) for pro ⁇ l (I) chains (COL1A1) .
  • Lanes 1 to 3 Cells treated with missense oligonucleotide MS3 (SEQ ID NO: 6) .
  • Lanes 4 to 6 Cells treated with the antisense oligonucleotide AS3 (SEQ ID NO: 5) .
  • Lanes 7 to 9 Control cells not treated with oligonucleotides. Three samples of cells were treated identically and analyzed separately in the lanes shown.
  • Figure 2 shows an assay of the steady-state levels of mRNAs from the exogenous (SEQ ID NO: 3) and endogenous (SEQ ID NO: 4) genes.
  • mRNA from the exogenous COL1A1 gene generates a band of 135 bases and mRNA from the endogenous C0L1A1 gene generates a band of 100 bases under the conditions of the experiment in which the same oligonucleotide primers are used to synthesize cDNAs from the mRNAs and cDMAs are amplified by the polymerase chain reaction followed by cleavage with a restriction endonuclease (BstNl) .
  • BstNl restriction endonuclease
  • Lanes 1-3 Treatment with 0.2 ⁇ M AS3 and 10 ⁇ g/ml lipofectin. Lanes 4-6: 0.2 ⁇ M MS3 and 10 ⁇ g/ml lipofectin. Lanes 7-9: 10 ⁇ g/ml lipofectin alone. Densitometry of the film demonstrated that AS3 decreased the level of the human mRNA to 80% of the value obtained with MS3
  • the present invention concerns oligonucleotides useful for inhibition of mutant collagen gene expression, and provides methods using these compounds for treatment of disorders caused by mutant collagen gene expression. It also concerns oligonucleotides useful for inhibition of expression of normal collagen genes to prevent excessive deposition of collagen in fibrotic conditions.
  • the invention provides an oligonucleotide substantially complementary to a mutant collagen nucleotide sequence or a normal collagen nucleotide sequence.
  • Nucleotide sequence refers to a polynucleotide formed from a series of joined nucleotide units.
  • substantially complementary refers to that amount of complementarity between the oligonucleotide and a collagen nucleotide sequence which allows for potentially stable interstrand hybridization and enables the oligonucleotide to inhibit the expression of the collagen gene.
  • Interstrand hybridization is the interaction between the oligonucleotide and the collagen nucleotide sequence.
  • the potential capability of forming a stable interstrand hybrid can be determined by those skilled in the art using methods known in the art, such as, for example, determination of the melting temperature for the hybrid (T m ) by mathematical modelling or empirical analysis, solid support nucleic acid hybridizations, or C 0 t analysis. (Marmur, J. and Doty, P., J. Mol . Biol . 1962, 5, 113) .
  • collagen nucleotide sequence refers to any nucleotide sequence derived from the a wild type or mutant collagen or procollagen gene, including, for example, DNA or RNA sequence, DNA sequence of the gene, any transcribed RNA sequence, RNA sequence of the pre-mRNA or RNA transcript, and DNA or RNA bound to protein.
  • Oligonucleotides useful for inhibition of mutant collagen gene expression may be selected by comparing a mutant collagen nucleotide sequence with a wild type collagen nucleotide sequence.
  • a region of the mutant collagen nucleotide sequence comprising at least one nucleotide difference from the wild type collagen nucleotide sequence may be selected as the target for the oligonucleotide.
  • An oligonucleotide complementary to this region is expected to be able to selectively hybridize to the mutant collagen nucleotide sequence but not the wild type nucleotide sequence.
  • neutral variations in the base sequence of an allele for a gene can be used as a target site for the oligonucleotide.
  • a panel of oligonucleotides to normal alleles can be used to inhibit expression of an allele that contains a neutral variation in sequence and a disease- causing mutation at a second site in the same allele, the use of a panel of oligonucleotides to normally functioning alleles will greatly reduce the number of specific oligonucleotides needed, since it will not be necessary to design a new oligonucleotide for each new mutation that is discovered in a given gene.
  • Oligonucleotides targeted to invariant sequences in collagen genes can be used to inhibit collagen synthesis in fibrotic conditions.
  • the oligonucleotide may be any length of sequence potentially capable of forming a stable hybrid with the mutant or normal collagen nucleotide sequence. The potential capability of forming a stable hybrid can be determined by those skilled in the art using methods known in the art. It is preferred that the length of the oligonucleotide be between 5 and 200 nucleotides. It is more preferred that the oligonucleotide be between 10 and 50 nucleotides in length. It is most preferred that the oligonucleotide be between 15 and 25 nucleotides in length.
  • wild type refers to the a natural, functional form of a collagen or procollagen nucleotide sequence. This includes, for example, natural, functional genes and transcripts of procollagen types I to XVI and to still undiscovered collagens that may be found in tissues of mammals.
  • Oligonucleotides substantially complementary to regions of the mutant nucleotide sequence that comprise a variation from the wild type nucleotide sequence of at least one point mutation, missense mutation, nonsense mutation, deletion, recombination, insertion or combinations of such mutations are preferred in the invention.
  • Normal invariant sequences are preferred to applications involving the inhibition of normal collagen synthesis and deposition.
  • a preferred embodiment of the invention is an oligonucleotide complementary to mutant or normal wild type nucleotide sequence of type I procollagen (C0L1A1 and COL1A2) , type II procollagen (C0L2A1) , type III procollagen (C0L3A1) or type IV collagen (COL4A1, COL4A2, COL4A3, C0L4A4 and COL4A5) . It is further preferred that the oligonucleotide be complementary to a nucleotide sequence derived or selected from a mammal, in particular, a human.
  • the oligonucleotides of the present invention may be oligodeoxyribonucleotides or oligoribonucleotides, including modified oligodeoxynucleotides and oligoribonucleotides. Moreover, the oligonucleotides of the invention may be comprised of combinations of deoxyribonucleotides and ribonucleotides.
  • oligonucleotides of the invention may also include modified subunits.
  • the invention may include phosphorothioate oligodeoxyribonucleotides.
  • the oligonucleotides of the invention be modified to increase stability and prevent intracellular and extracellular degradation. It is more preferred that the oligonucleotides of the invention be modified to increase their affinity for target sequences, and their transport to the appropriate cells and cell compartments when they are delivered into a mammal in a pharmaceutically active form.
  • Oligonucleotides of the invention may be synthesized by any method known in the art. It is preferred in the present invention that the oligonucleotides be prepared using synthetic chemical methods, such as, for example, phosphoramidite chemistry by sulfurization with tetraethylthiuram disulfide in acetonitrile. See, for example, Vu and Hirschbein, Tetrahedron Lett. 1991, 32, 30005-30008. Oligonucleotides of the invention may also be synthesized using in vi tro and in vivo transcription systems, such as transcription by T7 polymerase or expression vectors. Oligonucleotides synthesized using in vi tro and in vivo transcription systems may be modified via chemical methods known to those skilled in the art. Examples of such modifications include encapsulation in liposomes, or chemical linkage to steroids, antibodies, and cell receptors.
  • an oligonucleotide substantially complementary to a mutant or wild type collagen nucleotide sequence comprises a collagen gene expression control sequence.
  • the term "gene expression control sequence”, as used herein, denotes sequences that affect the level of expression a gene. Gene expression control sequences that affect the level of translation or the rate of RNA processing are preferred, but are the invention is not limited to sequences involved in these processes.
  • Gene expression control sequences useful in the invention include, for example, 5'- and 3' -splice junction sequence, splicing branchpoint sequence, small nuclear ribonucleoprotein binding site sequence (snRNP) , polyadenylation region sequence, translation initiation region sequence, transcript 5'- and 3' -untranslated region sequence, and sequence affecting RNA turnover.
  • the translation initiation site may include the Kozak sequence or other sequence embedding the start codon or adjacent to the start codon.
  • the 5' -splice junction sequence may include the Ul snRNP binding site or the 5' -splice junction octanucleotide consensus sequence.
  • the 5'-splice junction sequence may include sequences embedding the splice junction or adjacent to the splice junction.
  • the 3' -splice junction sequence may include sequences embedding the splice junction or adjacent to the splice junction.
  • the polyadenylation region sequence may include the AAUAAA consensus hexanucleotide, and active variants thereof, and sequences surrounding the cleavage site or adjacent to the cleavage site.
  • the target sequences for the oligonucleotides shall also include sequences that code for amino acid sequences in the proteins.
  • the oligonucleotides of the invention be antisense oligonucleotides. It is more preferred that the oligonucleotides of the invention be targeted to a collagen gene splice junction, in particular, a 5' -splice junction.
  • the term "splice junction" may encompass a 5'-splice junction sequence including the Ul snRNP binding site or the 5' -splice junction octanucleotide consensus sequence, or a 5' -splice junction sequence including sequences embedding the splice junction or adjacent to the splice junction.
  • splice junction may also denote a 3'-splice junction sequence including sequences embedding the splice junction or adjacent to the splice junction.
  • the splice junction of the invention may be, for example, an activated cryptic splice junction, a splice junction reconstituted at a deletion junction, or reconstituted by some other mutation, or a dominant splice junction within a mutated sequence milieu.
  • Another preferred method of the inventions is oligonucleotides targeted to coding sequences of the gene.
  • the invention further includes an oligonucleotide substantially complementary to a mutant collagen splice junction comprising SEQ ID NO: 18.
  • This consensus sequence is derived from a number of oligonucleotides tested which exhibit certain degrees of inhibitory activity on mutant collagen gene expression. See Tables 1 and 2.
  • mouse NIH 3T3 cells stably transfected with an internally deleted "mutant" construct of the human COL1A1 gene which encodes shortened pro ⁇ l(I) chains of type I procollagen were contacted with the oligonucleotides.
  • These oligonucleotides were synthesized using a region at the 3' end of exon 1 and the first two nucleotides of intron 1 of the exogenous human gene as a target. See Table 1. The target site was selected because the human gene contained 27 nucleotides in exon 1 that were not found in the corresponding endogenous mouse gene.
  • the target sequence of the most effective oligonucleotide tested was a 20 nucleotide stretch. This human collagen target sequence differed from the mouse sequence by nine nucleotides. The observed effects on expression were specific and ranged from 50 to 80% inhibition of the exogenous gene. Less than 10% inhibition of expression of the endogenous collagen gene or the fibronectin gene was observed. To further demonstrate the specificity of these oligonucleotides, missense or sense versions of the same oligonucleotide were tested. These oligonucleotides had essentially no effect on target gene expression. Also, the inhibition observed with the most effective oligonucleotide tested was reduced by introducing a single base change.
  • the invention also includes an oligonucleotide which comprises a sequence selected from the group of SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:ll, SEQ ID N0:12, SEQ ID NO:13, SEQ ID N0:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22 and SEQ ID NO:23.
  • oligonucleotides are complementary to the mutant 5' -splice junction sequence and effectively inhibit expression of the mutant exogenous gene. See Table 2.
  • Oligonucleotides having SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22 and SEQ ID NO:23 are particularly inhibitory. Oligonucleotides of the foregoing group are particularly useful as research reagents while not being limited to such use.
  • compositions for inhibiting mutant and normal collagen gene expression comprising an oligonucleotide of the invention, and a pharmaceutically acceptable carrier or diluent.
  • the invention further provides a preferred embodiments of pharmaceutical compositions comprising an oligonucleotide substantially complementary to a mutant or normal collagen gene expression control sequence, such as a collagen gene splice junction.
  • Pharmaceutical compositions comprising an oligonucleotide having SEQ ID NO: 18 are also included.
  • compositions comprising the oligonucleotides may be administered orally in solid dosage forms, such as capsules, tablets, and powders, or in liquid dosage forms, such as elixirs, syrups, and suspensions. They may also be administered parenterally in sterile liquid dosage forms as well as by inhalation or topical administration.
  • the dosage administered varies depending upon factors such as: pharmacodynamic characteristics; its mode and route of administration; age, health, and weight of the recipient; nature and extent of symptoms; kind of concurrent treatment; and frequency of treatment.
  • Effective dosages are those which are able to inhibit collagen protein production in cells at a level which eliminates or reduces the symptons or conditions associated with the collagen portein production.
  • the compounds may be formulated with a pharmaceutically acceptable topical carrier and the formulation to produce a creme, lotion or ointment for example.
  • the oligonucleotides of the invention may be mixed with a suitable carrier or diluent such as water, an oil, saline solution, aqueous dextrose, and other sugar solutions, glycols such as propylene glycol or polyethylene glycols, and lipids such as in liposomes or cationic lipids capable of binding nucleic acid.
  • a suitable carrier or diluent such as water, an oil, saline solution, aqueous dextrose, and other sugar solutions, glycols such as propylene glycol or polyethylene glycols, and lipids such as in liposomes or cationic lipids capable of binding nucleic acid.
  • a water soluble salt of an oligonucleotide of the invention may be used for parenteral administration.
  • Stabilizing agents, antioxidizing agents and preservatives may also be added.
  • Suitable antioxidizing agents include sodium bisulfite, sodium sulfite, and ascorbic acid, citric acid and its salts, and sodium EDTA.
  • Suitable preservatives include benzalkonium chloride, methyl- or propyl-paraben, and chlorbutanol.
  • Solutions for parenteral administration comprise preferably an oligonucleotide of the invention encapsulated in or bound to a cationic lipid.
  • lipofectin As a demonstration of the effectiveness of compositions comprising lipid, lipofectin was tested in a cell culture system. Lipofectin optimized inhibition for all of the antisense oligonucleotides tested. See Example 5, Tables 2 and 3, and Example 8, Table 4. The concentration of oligonucleotide required for effective inhibition were as low as 0.1 ⁇ M which is a physiologically acceptable concentration for mammalian therapeutic agents. In view of this observation it is expected that the oligonucleotides of the invention will be useful for treatment of mammals.
  • oligonucleotides of the invention may be administered by any method that produces contact of the oligonucleotide with the oligonucleotide's site of action in the body of a mammal including but not limited to oral, intravenous, and intraparenteral.
  • the oligonucleotides may be administered singly, or in combination with other compounds of the invention, other pharmaceutical compounds, or therapies.
  • the oligonucleotides are preferably administered with a pharmaceutically acceptable carrier or diluent selected on the basis of the selected route of administration and standard pharmaceutical practice.
  • the oligonucleotides of the invention are administered to mammals, preferably humans, in therapeutically effective amounts or concentrations which are effective to inhibit mutant collagen gene expression, or to treat diseases exhibiting mutant collagen gene expression.
  • the dosage administered in any particular instance will depend upon factors such as the pharraacodynamic characteristics of the particular oligonucleotide of the invention, and its mode and route of administration; age, health, and weight of the recipient; nature and extent of symptoms; kind of concurrent treatment, frequency of treatment, and the effect desired.
  • the invention provides methods of inhibiting mutant collagen gene expression which comprise contacting a cell comprising the mutant collagen gene with a mutant collagen gene expression inhibitory amount of an oligonucleotide substantially complementary to a mutant collagen nucleotide sequence or a neutral variation in the sequence of the same collagen allele containing the mutant sequence and not perfectly complementary to a wild type collagen nucleotide sequence or the target sequence in the allele for the wild type collagen allele.
  • the invention also includes a method whereby the contacting step comprises lipofectin as a carrier for the oligonucleotide.
  • the invention includes similar methods for inhibiting expression of wild type collagen genes.
  • the collagen nucleotide sequence comprises a collagen gene expression control sequence, such as a collagen gene splice junction, particularly a 5' -splice junction or a normal coding sequence.
  • the oligonucleotide comprises a sequence selected from the group of SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22 and SEQ ID NO:23. See Table 2. Methods comprising these oligonucleotides are particularly useful in research while not being limited to such use.
  • a further method of inhibiting mutant and wild type collagen gene expression is included wherein the oligonucleotide comprises SEQ ID NO: 18.
  • mouse NIH 3T3 cells stably expressing an internally deleted version of the human COLlAl gene were grown in cell culture.
  • the utility of the system was that it made it possible to assay directly changes in the expression of the exogenous human COLlAl relative to the expression of the endogenous mouse COLlAl.
  • the exogenous human COLlAl had an internal deletion of 40 exons (exons 6 to 45) engineered so that the mRNA and the pro ⁇ l(I) chains synthesized from the gene were less than half the size of the mRNA and pro ⁇ _l(I) chains synthesized from the mouse endogenous COLlAl gene.
  • specific inhibition of one gene relative to the other was readily assayed in the same sample of cells or tissues with techniques employed by those familiar in the art.
  • RNA and protein analyses demonstrated that contacting cells expressing the mutant human collagen gene with certain oligonucleotides of the invention using the above method significantly inhibited the mutant gene expression. See Example 5, Tables 2 and 3, and Example 8, Table 4. Maximal inhibition with AS3 (SEQ ID NO: 5) was observed in about 20 hours. See Figure 3. The inhibition began after 8 hours and persisted for at least 30 hours.
  • the mutated human COLlAl gene used in these experiments with cells was modeled after a mutated COLlAl gene shown to cause a lethal form of osteogenesis imperfecta. Williams, C.J. and Prockop, D.J., J. Biol . Chem. 1983, 258, 5915-5921; and Olsen et al. , J. Biol . Che . 1991, 266, 1117- 1121.
  • the same mutated human COLlAl gene was used to prepare transgenic mice. Lines of transgenic mice expressing high levels of the gene were shown to develop fractures of bones similar to those seen in children with osteogenesis imperfecta. Khillan, J.S. et al. , J. Biol . Chem.
  • oligonucleotides that inhibit the expression of the mutated human COLlAl gene in cell culture offer the potential of inhibiting expression of the same mutated human COLlAl gene in the transgenic mice and thereby rescuing the phenotype of fragile bones in the transgenic mice.
  • Successful testing of the oligonucleotides in the transgenic mice would offer the prospect of using the same or similar oligonculeotides to treat or prevent fragile bones in children with osteogenesis imperfecta.
  • the same or similar oligonucleotides may be useful in treating or preventing osteoporosis.
  • the same cells and transgenic mice expressing the mutated human COLlAl gene can be used to develop oligonucleotides to specifically inhibit expression of normal human COLlAl genes by targeting either invariant sequences in the human gene or regions that contain neutral variations in normal alleles of genes.
  • the oligonucleotides of the invention will be capable of reaching their intracellular target to affect inhibition of mutant collagen gene expression.
  • the invention therefore provides methods of inhibiting mutant and wild type collagen gene expression which comprise contacting at least one element of gene expression machinery with a collagen gene expression inhibitory amount of an oligonucleotide.
  • the elements of the gene expression machinery may comprise any nucleotide sequence of a gene, the nucleotide sequence of spliced mRNAs transcribed from a gene, unspliced RNAs and partially spliced RNAs transcribed from a gene, DNA- RNA hybrids comprising sequence derived from a gene, such as in actively transcribing genes, RNA transcribed from a gene bound to protein, and any molecule or structure known in the art to be involved in gene expression.
  • the oligonucleotides of the invention will be capable of inhibiting collagen gene expression in cells in vivo and in vi tro, including, for example, individual cells, cells comprising tissues, cells comprising organs, and cells comprising organisms.
  • the inhibitory effect of an oligonucleotide ranges from the lowest statistically significant inhibitory level to about 100% inhibition.
  • one skilled in the art will be able to design or select oligonucleotides which are appropriate for the degree of inhibition which is needed for the desired purpose without undue experimentation.
  • the invention includes a method for treating a disease exhibiting mutant collagen gene expression which comprises administering to a mammal suffering from a disease caused by expression of a mutant collagen gene, an inhibitory amount of an oligonucleotide substantially complementary to a mutant collagen nucleotide sequence and not perfectly complementary to mutant collagen nucleotide sequence.
  • the inhibitory oligonucleotide can be substantially complementary to a neutral sequence variation found in the same collagen allele containing the mutation but not substantially complementary to the same target site in the second allele for the same collagen in the same individual.
  • the methods of the invention also include administering to a mammal suffering from a fibrotic condition, an inhibitory amount of an oligonucleotide substantially complementary to an invariant sequence in the wild type sequence of a neutral variant sequence in the wild type gene to inhibit, specifically, expression of the gene and, thereby, prevent deleterious deposition of collagen in tissues.
  • the methods of the invention also include the delivery of oligonucleotides to certain regions of the mammal being treated, such as, for example, by specific and targeted delivery of the oligonucleotides to certain organs or tissues, including bolus delivery and immunological targeting.
  • pharmaceutical preparations comprising the oligonucleotides may be injected directly into a desired bodily site.
  • antibodies may be bound to the oligonucleotides or oligonucleotide-lipid complexes to direct the oligonucleotides to cells expressing certain antigens, such as virally infected cells. It is believed that the methods of the invention for treating disease are particularly useful in the treatment of human diseases of collagen, including, for example, osteogenesis imperfecta, chondrodysplasia and Ehlers-Danlos syndrome type IV. It is also believed they will be useful in treating subsets of patients with specific types of osteoporosis, osteoarthritis and aneurysms.
  • fibrotic conditions such as liver cirrhosis, pulmonary fibrosis, scleroderma, hypertrophic scar formation and keloids. It is also believed they will be useful in treating normal individuals to prevent fibrotic scarring following trauma or surgical procedures.
  • the invention provides a preferred method of treatment wherein the mutant collagen nucleotide sequence comprises a collagen gene expression control sequence, such as a collagen gene splice junction.
  • a collagen gene expression control sequence such as a collagen gene splice junction.
  • Another method for treating a disease exhibiting mutant collagen gene expression is included wherein the oligonucleotide comprises SEQ ID NO: 18.
  • the usefulness of the oligonucleotides of the invention in the treatment of mammals can be seen from the concentrations of oligonucleotide sufficient to inhibit gene expression.
  • concentration of oligonucleotide required for effective inhibition was as low as 0.1 ⁇ M. See Example 8, Table 4.
  • it is expected that the same or similar oligonucleotides in a pharmaceutically acceptable carrier will be useful to rescue the phenotype of fragile bones in transgenic mice expressing the same internally deleted gene. It is also expected that these oligonucleotides will be useful to inhibit the expression of mutant collagen genes in collagen disorders of humans.
  • oligonucleotide is specifically targeted to an invariant region of the wild type human COLlAl gene, it is also expected that it will be useful in treating fibrotic conditions in man and other mammals.
  • Phosphorothioate oligodeoxynucleotides were synthesized via phosphoramidite chemistry by sulfurization with tetraethylthiuram disulfide in acetonitrile. See Vu and Hirschbein, Tetrahedron Lett . 1991, 32, 3005-3008.
  • DMEM Dulbecco's modified Eagle's medium
  • GEBCO BRL Gaithersberg, MD
  • Oligonucleotides dissolved in distilled water were then added as a 2OX stock solution and incubated for 4 hours at 37°C.
  • About 0.7 ml of DMEM containing 14% calf serum previously heat inactivated at 56°C for 1 hour and 400 ⁇ g/ml of Geneticin were added. The cells were then incubated at 37°C for the additional times indicated.
  • Example 3 Protein Analysis
  • oligonucleotides were washed two times in DMEM and solubilized in 0.1 ml of lysis buffer consisting of 1% SDS; 1% sodium deoxycholate; 0.1% Triton X-100; 10 mM EDTA; 0.5 units of aprotinin (Sigma, St. Louis, MO) per ml; 3% /3-mercaptoethanol; and phosphate buffered saline (PBS) adjusted to pH 7.4.
  • lysis buffer consisting of 1% SDS; 1% sodium deoxycholate; 0.1% Triton X-100; 10 mM EDTA; 0.5 units of aprotinin (Sigma, St. Louis, MO) per ml; 3% /3-mercaptoethanol; and phosphate buffered saline (PBS) adjusted to pH 7.4.
  • sample loading buffer 0.6 M Tris-HCl buffer, pH 6.8; 50% glycerol; 1% SDS; 0.012% bromphenol blue.
  • the lysate was then heated for 5 minutes at 94°C and 10 ⁇ l of the sample was electrophoresed on a 7.0% SDS polyacrylamide gel. Proteins were electrophoretically transferred to nitrocellulose filters (Schleicher and Schuell, Keene, NH) and reacted with an antibody against a synthetic peptide corresponding to the last 21 amino acids of human pro ⁇ l (I) chain of type I procollagen. The antibody recognized both the human and the mouse COOH- terminal propeptide of the pro ⁇ .1 (I) chain. Olsen, A. S., J. Biol . Chem. 1991, 266, 1117-1121.
  • pro ⁇ .1 (I) bands were detected by reaction with a goat anti-rabbit antibody coupled to 125I (Dupont-NEN, Boston, MA) and subsequent autoradiography. Relative amounts of protein from the endogenous and exogenous COLlAl genes were then assayed by using a laser densitometer (LKB, Ultroscan XL, Piscataway, NJ) .
  • LLB laser densitometer
  • RNA assays total cellular RNA was isolated from tissues using acidic guanidine thiocyanate-phenol-chloroform extraction. Chomczynski, P., and Sacchi, M. , Anat. Biochem. 1987, 162, 156-159. The ratio of mRNA from exogenous and endogenous genes was measured by a quantitative polymerase chain reaction (PCR) assay. Primers for reverse transcription and polymerase chain reaction were designed to be complementary to identical sequence in human and mouse pro ⁇ .1 (I) mRNA. Mooslehner, K. , and Habers, K. , Nucl . Acids Res . 1988, 16, 773; Westefflausen, A., Matrix. Col . Rel . Res . 1991, 11, 375-379.
  • PCR polymerase chain reaction
  • primer BS31 (5'- TTGGCCCTGTCTGCCT-3') (SEQ ID NO: 1) and 32 P-labeled primer BS32
  • test system employed mouse NIH 3T3 cells stably transfected with an internally deleted construct of the human gene for the pro ⁇ .l(I) chains of type I procollagen (COLlAl). See Prockop, D. J., ⁇ J. Biol . Chem. 1990, 265, 15349-15352.
  • a series of modified oligonucleotides were synthesized using a region at the 3' end of exon 1 and the first two nucleotides of intron 1 of the exogenous gene as a target (Table 1) .
  • SEQ ID NO: 6 contains the same content in A, C, G and T as AS3 (SEQ ID NO: 5) , but in a random order.
  • S3 (SEQ ID NO: 7) is the sense version of AS 3 (SEQ ID NO: 5) .
  • oligonucleotides tested were effective in inhibiting expression of either the exogenous or the endogenous gene when the oligonucleotide was administered without any carrier, even at concentrations up to 25 ⁇ M.
  • several of the oligonucleotides designed as antisense inhibitors of the exogenous gene were effective when administered with 10 ⁇ g/ml lipofectin which increases the uptake of nucleic acid, Chiang, M.-Y. et al., J. Biol . Chem. 1991, 266, 18162-18171.
  • the small degrees of inhibition seen with MS3 (SEQ ID NO: 6) and S3 were not consistently observed in all experiments.
  • the relative effectiveness of the oligonucleotides was more apparent when the values were compared to the variable values seen with the missense oligonucleotide MS3 (SEQ ID NO: 6) .
  • AS3 SEQ ID NO: 5
  • AS7 SEQ ID NO: 8
  • AS16 SEQ ID NO: 16
  • the mRNAs from cells were transcribed into single-stranded cDNAs using an oligonucleotide that primed both the mRNA for the human and mouse prootl(I) chain.
  • the single-stranded cDNA was then amplified by PCR using a single set of primers with one of the primers labeled with 32P.
  • the antisense oligonucleotide AS3 selectively decreased the steady-state level of mRNA for pro ⁇ l (I) chains from the exogenous gene to about 50% of the control value ( Figure 2) .
  • the relative expression at the protein level was also decreased by about 50%.
  • Example 9 Inhibition of hepatic fibrosis in rats produced by carbon tetrachloride and dimethylnitrosamine Cirrhosis of the liver is a potentially lethal condition in which normal liver tissue is gradually replaced by collagen fibers following injury to liver by viruses, alcohol or toxic chemicals. The development of hepatic fibrosis is frequently studied experimentally in rats by administering carbon tetrachloride or dimethylnitrosamine to the rats.
  • mice Female Sprague-Dawley rats, 8 weeks old, were used for the induction of liver fibrosis.
  • the initial body weight of the animals was approximately 200 gm.
  • the animals were maintained on a normal diet with free access to water and a 12-hour light and dark cycle.
  • the CC1 4 was mixed with an equal volume of mineral oil and injected intraperitoneally at doses of 0.1 ml/100 gm body weight, twice a week for 42 days. The animals were killed at regular intervals over a period of 42 days. Five control animals and seven test animals were injected at the same time.
  • DN dimethylnitrosamine
  • doses of 1 ⁇ l diluted 1:100 with 0.15 mol/L NaCl per 100 gm body weight were administered.
  • the injections were made on the first 3 days of each week over a period of 28 days.
  • Treated animals were killed on days 7, 14, 21 or 28.
  • Control animals were killed at the same time.
  • Each group killed on a given day consisted of five control and seven treated animals.
  • Rats were anesthetized with diethylether and the livers were immediately removed and frozen in liquid nitrogen. The livers were stored frozen at -70° until analyzed. Samples of serum were removed at the same time and stored frozen.
  • the livers were thawed and homogenized in a teflon and glass homogenizer. A portion of the homogenate was taken for protein measurements. A portion of the homogenate was taken for protein measurements. A portion of the homogenate was used for assays of hydroxyproline alter hydrolysis in 6 mol/L HC1 and dansyl modification.
  • the filters were washed twice in 1 X standard saline citrate and 0.1% SDS at room temperature for 15 minutes and then twice in 0.1 X standard saline citrate and 0.1% SDS at 55°C for 30 minutes.
  • the cDNA probes were for the human pro ⁇ ld) chain, the human pro ⁇ l(III) chain, the mouse onl(IV) chain, the mouse ⁇ 2(IV) chain, and the mouse laminin B2 chain.
  • thin slices of liver were fixed in 10% neutral-buffered formalin and embedded in paraffin.
  • Sections were cut at 5 ⁇ m thickness, stained with hematoxylin and eosin and the Masson trichrome stain for collagen, and subjected to silver impregnation for the demonstration of reticulin fibers.
  • Microscopic evaluation of fibrosis, liver cell damage, inflammation and ductular cell proliferation was performed without knowledge of the source of the specimen. The various parameters were graded from 0 to +3 in order of increasing severity. As indicated in Table 5, treatment of rats with DMN or CC1 4 increased the total content in liver of collagen hydroxyproline. To confirm the fibrotic effects of the two agents, livers from the rats were examined by light microscopy, without knowledge of the source of the specimen. As indicated in Table 6, the expected fibrotic changes were observed.
  • oligonucleotide targeted to one or more of the collagen genes would provide an effective method of limiting the fibrotic response.
  • the experiments in rats provide a useful system for testing the effectiveness of the oligonucleotides. For example, administering an oligonucleotide that inhibits expression of the COLlAl gene for type I procollagen should inhibit the increase in mRNA for the ⁇ l(I) chain for type I collagen and increase in collagen liver hydroxyproline seen in Table 5. Therefore, the oligonucleotide should prevent the fibrosis seen in Table 6. Obtaining such results in rats should provide part of the information necessary to test the effectiveness of the same oligonucleotide for preventing liver fibrosis and cirrhosis and other fibrotic conditions in man and other mammals.
  • Mutations in the two genes for type I procollagen cause osteogenesis imperfecta and a subset of osteoporosis
  • mutations in the gene for type II procollagen cause chondrodysplasias and some forms of osteoarthritis
  • mutations in the gene for type III procollagen cause Ehlers-Danlos syndrome type IV and a subset of aneurysms.
  • mutations in the genes for type IV collagen cause the renal disease and other features of the Alport syndrome. Mutations in type IV collagen may also cause glomerulonephrosis and more common renal diseases. Examination of the collagen mutations causing these diseases, however, has demonstrated that most unrelated probands and families have a different mutation in the same collagen gene.
  • neutral sequence variations in the large cluster of genes have been used to define specific haplotypes of the gene, i.e., patterns of neutral variations in and around the genes that can be used to distinguish the gene cluster of one chromosome from another.
  • Orkin, S . The Molecular Basis of Blood Diseases, G. Stamatoyannopoulos, A.W. Nieehuis, P. Leder and P.W. Majerus, Eds., W.B. Saunders, Philadelphia, 1987, p 166. It is very likely that the neutral sequence variants seen in human type II procollagen gene define specific neutral haplotypes of the gene.
  • the 25 neutral variations shown in Table 7 probably occur within specific patterns in alleles of the gene so that they define many fewer than 25 different alleles.
  • the presence of such neutral variations provides specific target sites for oligonucleotides to inhibit expression of specific alleles of type II procollagen gene. Therefore, if a disease in a proband or family is shown to be caused by the mutation in a specific allele, an oligonucleotide targeted to a neutral sequence variation in the same allele will be effective in inhibiting expression of the allele. Therefore, if the 25 neutral variations shown in Table 7 define five specific alleles of collagen II gene, five specific oligonucleotides will be adequate to specifically inhibit expression of a large number of different mutations that may occur in the same allele.
  • Values are mean relative to controls ⁇ S.D. Values for liver hydroxyproline were first calculated as milligrams of hydroxyproline per gram wet weight of liver and then as percent of control values.
  • Position for exon is designated without a ⁇ sign.
  • Position in intron is designated with ⁇ a"-" sign if the sequence variant is located 5' to the next exon and with a ⁇ +" sign if the sequence variant is located 3' to the preceding exon.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Genetics & Genomics (AREA)
  • Public Health (AREA)
  • Animal Behavior & Ethology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Veterinary Medicine (AREA)
  • Medicinal Chemistry (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • General Chemical & Material Sciences (AREA)
  • Molecular Biology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Biomedical Technology (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Biochemistry (AREA)
  • Microbiology (AREA)
  • Plant Pathology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Dermatology (AREA)
  • Pulmonology (AREA)
  • Epidemiology (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Saccharide Compounds (AREA)

Abstract

L'invention concerne un oligonucléotide pratiquement complémentaire d'une séquence de nucléotides de collagène mutants et pas parfaitement complémentaire d'une séquence de nucléotides de collagène sauvages. L'invention porte également sur un procédé de sélection et de préparation de ce type d'oligonucléotide, ainsi que sur des procédés de traitement de mammifères souffrant de maladies présentant une expression des gènes de collagène mutants qui consistent à utiliser l'oligonucléotide selon l'invention pour inhiber l'expression des gènes de collagène mutants.
PCT/US1993/010756 1992-11-09 1993-11-09 Oligonucleotides antisens utilises pour inhiber l'expression de genes de collagene ayant subis une mutation et de type sauvage WO1994011494A1 (fr)

Priority Applications (3)

Application Number Priority Date Filing Date Title
US08/432,158 US5861502A (en) 1992-11-09 1993-11-09 Antisense oligonucleotides to inhibit expression of mutated and wild type genes for collagen
EP94901327A EP0674705A4 (fr) 1992-11-09 1993-11-09 Oligonucleotides antisens utilises pour inhiber l'expression de genes de collagene ayant subis une mutation et de type sauvage.
JP6512264A JPH08503366A (ja) 1992-11-09 1993-11-09 コラーゲンの突然変異および野生型遺伝子の発現を阻害するアンチセンスオリゴヌクレオチド

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US97333292A 1992-11-09 1992-11-09
US07/973,332 1992-11-09

Publications (1)

Publication Number Publication Date
WO1994011494A1 true WO1994011494A1 (fr) 1994-05-26

Family

ID=25520770

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1993/010756 WO1994011494A1 (fr) 1992-11-09 1993-11-09 Oligonucleotides antisens utilises pour inhiber l'expression de genes de collagene ayant subis une mutation et de type sauvage

Country Status (4)

Country Link
EP (1) EP0674705A4 (fr)
JP (1) JPH08503366A (fr)
CA (1) CA2148687A1 (fr)
WO (1) WO1994011494A1 (fr)

Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997011169A3 (fr) * 1995-09-21 1997-06-12 Trinity College Dublin Strategie permettant de supprimer l'expression d'un gene endogene en utilisant des composes capables de se lier aux regions non codantes d'un gene a supprimer
WO1997032024A1 (fr) * 1996-03-01 1997-09-04 Provost, Fellows And Scholars Of The College Of The Holy And Undivided Trinity Of Queen Elizabeth Near Dublin Suppression d'alleles
WO1997037014A1 (fr) * 1996-04-02 1997-10-09 Provost, Fellows And Scholars Of The College Of The Holy And Undivided Trinity Of Queen Elizabeth Near Dublin Suppression et remplacement genetiques
US5780611A (en) * 1995-09-15 1998-07-14 Ramareddy Venkata Guntaka Oligomers which inhibit expression of collagen genes
US5808037A (en) * 1995-09-15 1998-09-15 Ramareddy Venkata Guntaka Oligomers which inhibit expression of collagen genes
WO1998041648A3 (fr) * 1997-03-20 1999-04-29 Variagenics Inc Genes cibles pour medicaments specifiques d'alleles
WO2000008213A1 (fr) * 1998-08-07 2000-02-17 Guntaka Ramareddy V Oligomeres inhibant l'expression de genes de collagene
US6200754B1 (en) 1998-03-19 2001-03-13 Variagenics, Inc. Inhibitors of alternative alleles of genes encoding products that mediate cell response to environmental changes
WO2001044455A3 (fr) * 1999-12-15 2002-01-10 Astrazeneca Ab Oligonucleotides antisens
EP0948513A4 (fr) * 1996-10-31 2004-12-29 Univ Johns Hopkins Med Produit de recombinaison pour l'administration d'acides nucleiques antisens et ses procedes d'utilisation
US6939712B1 (en) * 1998-12-29 2005-09-06 Impedagen, Llc Muting gene activity using a transgenic nucleic acid
US8551970B2 (en) 1996-04-02 2013-10-08 Optigen Patents Limited Genetic suppression and replacement

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CA2006008C (fr) * 1988-12-20 2000-02-15 Donald J. Kessler Methode pour obtenir des oligonucleotides synthetiques qui se lient specifiquement a des sites cibles sur des molecules d'adn double brin, en formant un triplex colineaire; methode d'utilisation de ces oligonucleotides synthetiques
JPH07500820A (ja) * 1991-09-05 1995-01-26 ユニバーシティ オブ コネティカット ポリヌクレオチド又はオリゴヌクレオチドの細胞への標的を定めた送達

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
Annu. Rev. Biochem., Vol. 59, issued 1990, VUORIO et al., "The Family of Collagen Genes", pages 837-872, see the entire document. *
Chemical Reviews, Vol. 90, Number 4, issued June 1990, UHLMANN et al., "Antisense Oligonucleotides: A New Therapeutic Principle", pages 544-579, see entire document. *
See also references of EP0674705A4 *
The American Journal of Human Genetics, Vol. 51, No. 4, Abstract 1505, issued October 1992, HORTON et al., "Antisense Regulation of Human Type II Collagen Synthesis", see entire Abstract. *

Cited By (21)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5780611A (en) * 1995-09-15 1998-07-14 Ramareddy Venkata Guntaka Oligomers which inhibit expression of collagen genes
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
US6713457B2 (en) 1995-09-21 2004-03-30 Gwenyth Jane Farrar Strategy for suppressing the expression of an endogeneous gene by using compounds that are able to bind to the non-coding regions of the gene to be suppressed
WO1997011169A3 (fr) * 1995-09-21 1997-06-12 Trinity College Dublin Strategie permettant de supprimer l'expression d'un gene endogene en utilisant des composes capables de se lier aux regions non codantes d'un gene a supprimer
WO1997032024A1 (fr) * 1996-03-01 1997-09-04 Provost, Fellows And Scholars Of The College Of The Holy And Undivided Trinity Of Queen Elizabeth Near Dublin Suppression d'alleles
AU728506B2 (en) * 1996-03-01 2001-01-11 Optigen Patents Limited Allele suppression
WO1997037014A1 (fr) * 1996-04-02 1997-10-09 Provost, Fellows And Scholars Of The College Of The Holy And Undivided Trinity Of Queen Elizabeth Near Dublin Suppression et remplacement genetiques
US8551970B2 (en) 1996-04-02 2013-10-08 Optigen Patents Limited Genetic suppression and replacement
US7138378B1 (en) 1996-04-02 2006-11-21 Optigen Patents Limited Genetic suppression and replacement
EP0948513A4 (fr) * 1996-10-31 2004-12-29 Univ Johns Hopkins Med Produit de recombinaison pour l'administration d'acides nucleiques antisens et ses procedes d'utilisation
WO1998041648A3 (fr) * 1997-03-20 1999-04-29 Variagenics Inc Genes cibles pour medicaments specifiques d'alleles
US6200754B1 (en) 1998-03-19 2001-03-13 Variagenics, Inc. Inhibitors of alternative alleles of genes encoding products that mediate cell response to environmental changes
WO2000008213A1 (fr) * 1998-08-07 2000-02-17 Guntaka Ramareddy V Oligomeres inhibant l'expression de genes de collagene
US6939712B1 (en) * 1998-12-29 2005-09-06 Impedagen, Llc Muting gene activity using a transgenic nucleic acid
WO2001044455A3 (fr) * 1999-12-15 2002-01-10 Astrazeneca Ab Oligonucleotides antisens
EP1698695A2 (fr) 1999-12-15 2006-09-06 Nath, Rahul Kumar, M.D. Oligonucléotides antisens pour l'inhibition de l'expression du procollagène du type I
US7173122B2 (en) 1999-12-15 2007-02-06 Rahul Kumar Nath Antisense oligonucleotides to type I procollagen
EP1698695A3 (fr) * 1999-12-15 2007-02-14 Nath, Rahul Kumar, M.D. Oligonucléotides antisens pour l'inhibition de l'expression du procollagène du type I
EP2025752A2 (fr) 1999-12-15 2009-02-18 Nath, Rahul Kumar, M.D. Oligonucléotides antisens pour inhiber l'expression de procollagène de type 1
EP2025752A3 (fr) * 1999-12-15 2009-08-12 Nath, Rahul Kumar, M.D. Oligonucléotides antisens pour inhiber l'expression de procollagène de type 1

Also Published As

Publication number Publication date
EP0674705A4 (fr) 1997-11-26
CA2148687A1 (fr) 1994-05-26
EP0674705A1 (fr) 1995-10-04
JPH08503366A (ja) 1996-04-16

Similar Documents

Publication Publication Date Title
JP7423015B2 (ja) キメラ２重鎖核酸
US7662948B2 (en) Antisense oligonucleotides against VR1
US5747470A (en) Method for inhibiting cellular proliferation using antisense oligonucleotides to gp130 mRNA
US6005095A (en) Antisense transcript associated to tumor cells having a T(14;18) translocation and oligodeoxynucleotides useful in the diagnosis and treatment of said tumor cells
JP4846965B2 (ja) 真核細胞におけるエキソンスキッピングの誘導
KR100316205B1 (ko) 평활근세포의증식을조절하기위한c-myc의안티센스억제
JP3054745B2 (ja) ｒａｆ遺伝子発現のアンチセンスオリゴヌクレオチド調節
AU687001B2 (en) Method and reagent for inhibiting cancer development
EP2516647B1 (fr) Molécule pour le traitement d'un trouble inflammatoire
Laptev et al. Specific inhibition of expression of a human collagen gene (COL1A1) with modified antisense oligonucleotides. The most effective target sites are clustered in double-stranded regions of the predicted secondary structure for the mRNA
JPH09507502A (ja) ｒａｆ遺伝子発現のアンチセンスオリゴヌクレオチド調節
US5866699A (en) Oligonucleotides with anti-MDR-1 gene activity
EP0674705A1 (fr) Oligonucleotides antisens utilises pour inhiber l'expression de genes de collagene ayant subis une mutation et de type sauvage
US5683987A (en) Therapeutic oligonucleotides targeting the human MDR1 and MRP genes
US5861502A (en) Antisense oligonucleotides to inhibit expression of mutated and wild type genes for collagen
EP0625194B1 (fr) Intensification de l'activite catalytique des ribozymes en utilisant un oligonucleotide de facilitation voisin
US5872007A (en) CAPL-specific oligonucleotides and methods of inhibiting metastatic cancer
KR20200014320A (ko) Apcs의 발현을 억제하는 핵산
CA2248932A1 (fr) Oligonucleotides cibles sur l'arnm de l'angiotensinogene
US5780612A (en) Oligonucleotides specific for cytokine signal transducer gp130 mRNA
US6544755B1 (en) Method and reagent for treatment of diseases by expression of the c-Myc gene
EP0747480A1 (fr) Oligonucléotides spécifiques de l'ARNm du transducteur de signal pour les cytokines gp130
CN116376906A (zh) 靶向人Lpa(APO(a))mRNA的反义寡核苷酸和siRNA及其应用
WO2004050674A2 (fr) Procedes et materiaux de modulation des trpm2
EP1470145A2 (fr) Methodes et materiels destines a la modulation de p2x2

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): CA JP US

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): AT BE CH DE DK ES FR GB GR IE IT LU MC NL PT SE

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

Ref document number: 2148687

Country of ref document: CA

WWE Wipo information: entry into national phase

Ref document number: 1994901327

Country of ref document: EP

WWE Wipo information: entry into national phase

Ref document number: 08432158

Country of ref document: US

WWP Wipo information: published in national office

Ref document number: 1994901327

Country of ref document: EP

WWW Wipo information: withdrawn in national office

Ref document number: 1994901327

Country of ref document: EP