WO1993008845A1 - Traitement par application localisee d'oligonucleotides - Google Patents

Traitement par application localisee d'oligonucleotides Download PDF

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Publication number
WO1993008845A1
WO1993008845A1 PCT/US1992/009626 US9209626W WO9308845A1 WO 1993008845 A1 WO1993008845 A1 WO 1993008845A1 US 9209626 W US9209626 W US 9209626W WO 9308845 A1 WO9308845 A1 WO 9308845A1
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oligonucleotide
oligonucleotides
antisense
myb
sequence
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PCT/US1992/009626
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English (en)
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Robert D. Rosenberg
Michael Simons
Elazer Edelman
Robert S. Langer
Jean-Luc Dekeyser
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Massachusetts Institute Of Technology
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A61K9/0024—Solid, semi-solid or solidifying implants, which are implanted or injected in body tissue
    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1135—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against oncogenes or tumor suppressor genes
    • 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

  • oligonucleotides are capable of inhibiting the replication of certain viruses in tissue culture systems.
  • Zamecnik and Stephenson Proc. Natl. Acad. Sci. U.S.A. ,75:280-284 (1978)
  • Zamecnik et al. Proc. Natl. Acad. Sci. U.S.A.
  • oligonucleotides are subject to being degraded or inactivated by cellular endogenous nucleases.
  • modified oligonucleotides e.g., having altered internucleotide linkages, in which the naturally occurring phosphodiester linkages have been replaced with another linkage.
  • Agrawal et al., Proc Natl. Acad. Sci. U.S.A.,85:7079-7083 (1988) showed increased inhibition in tissue culture of HIV-1 using oligonucleotide phosphoramidates and phosphorothioates. Sarin et al., Proc. Natl.
  • Oligonucleotides having artificial linkages have been shown to be resistant to degradation in vivo.
  • Shaw et al. in Nucleic Acids Res. ,19:747-750 (1991), report that otherwise unmodified oligonucleotides become more resistant to nucleases in vivo when they are blocked at the 3' end by certain capping structures and that uncapped oligonucleotide phosphorothioates are not degraded in vivo.
  • oligonucleotides While antisense oligonucleotides have been shown to be capable of interfering selectively with protein synthesis, and significant progress has been made on improving their intracellular stability, the problem remains that oligonucleotides must reach their intended intracellular site of action in the body in order to be effective. Where the intended therapeutic effect is a systemic one, oligonucleotides may be administered systemically. However, when it is necessary or desirable to administer the oligonucleotide to a specific region within the body, systemic administration typically will be unsatisfactory. This is especially true when the target mRNA is present in normal cells as well as in the target tissue, and when antisense mRNA binding in normal cells induces unwanted physiological effects. Stated differently, the dosage of antisense oligonucleotide administered systemically that is sufficient to have the desired effect locally may be toxic to the patient.
  • An example of a treatment strategy which could greatly benefit from development of a method of limiting the effect of antisense oligonucleotide to a target tissue is the inhibition of smooth muscle cell proliferation which leads to restenosis following vascular trauma.
  • Smooth muscle cell proliferation is a poorly understood process that plays a major role in a number of pathological states including atherosclerosis and hypertension. It is the leading cause of long-term failure of coronary and peripheral angioplasty as well as of coronary bypass grafts.
  • Vascular smooth muscle cells in adult animals display a well defined phenotype characterized by an abundance of contractile proteins, primarily smooth muscle actin and myosins, as reviewed by S.M. Schwartz, G.R. Campbell, J.H. Campbell, Circ.
  • a nuclear oncogene c-myb may play an important role in these changes.
  • the oncogene is homologous to the transforming gene of the avia myeloblastosis virus. Although considered originally to be expressed only in hematopoietic cells, c-myb has been shown to be present in chick embryo fibroblasts as well as in proliferating SMCs. C.B. Thompson, et al. Nature, 319:374 (1986); C.F. Reilly, et al. J. Biol Chem., 264:6990 (1989). The human c-myb gene has been isolated, cloned and sequenced. Majello et al., Proc. Natl. Acad. Sci.
  • c-myb is growth-dependent. It is present in a low level in quiescent cells but increases rapidly as cells begin to proliferate and peaks near the late G phase of the cell cycle. C.F. Reilly, et al. J. Biol. Chem., 264:6990 (1989). Furthermore, expression of c-myb appears to correlate with the differentiation state of the cell. Myeloid erythroleukemia cells have been induced to differentiate and thereby decrease c-myb expression.
  • Heparin as well as closely related heparin proteoglycans, can block smooth muscle cell proliferation .in vivo as well as jLn vitro. A.W. Clowes, M.J. Karnovsky Nature, 265:625 (1977); C.R.
  • heparin may exert its antiproliferative action by its effect on c-myb. It is an object of the present invention to provide a method for delivery of oligonucleotides to a specific locus in vivo, and thereby to provide localized inhibition of expression of viral genes, oncogenes and genes encoding proteins involved in disease or other pathologic conditions. Summary of the Invention
  • the present invention relates to a method for inhibiting translation or transcription of a target nucleic acid sequence preferentially at a locus in vivo.
  • the invention involves application directly to the target tissue through a surgical or catheterization procedure of specific oligonucleotides having a nucleotide sequence complementary to at least a portion of the target nucleic acid, i.e., antisense oligonucleotides.
  • the oligonucleotides are preferably antisense sequences specific for the messenger RNA (mRNA) transcribed from the gene whose expression is to be inhibited.
  • the antisense oligonucleotides hybridize with the target mRNA thereby preventing its translation into the encoded protein.
  • the present method prevents the protein encoded by a selected gene from being expressed. Furthermore, animal experiments have demonstrated dramatic local therapeutic effects in vivo.
  • the present oligonucleotides preferably are modified to render them resistant to degradation and/or extension by cellular nucleases or other enzymes present in vivo. This can be accomplished by methods known in the art, e.g., by incorporating one or more internal artificial internucleotide linkages, such as replacing the phosphate in the linkage with sulfur, and/or by blocking the 3' end of the oligonucleotide with capping structures.
  • Oligonucleotides of the present invention are preferably between about 14 and 38 nucleotides in length, more preferahly between 15 and 30 nucleotides.
  • the oligonucleotides are applied locally in order to suppress expression of the protein of choice in a circumscribed area.
  • the antisense oligonucleotide is applied to the surface of the tissue at the locus disposed within a biocompatible matrix or carrier.
  • the matrix or carrier can be a hydrogel material such as a poly(propylene oxide-ethylene oxide) gel, e.g., one which is liquid at or below room temperature, and is a gel at body temperature and above.
  • the oligonucleotides are mixed with the hydrogel material, and the mixture is applied to the desired location during surgery or by catheter.
  • the oligonucleotides also can be applied in solution by liquefying the gel, i.e., by cooling, and are retained at the area of application as the gel solidifies.
  • Other carriers which can be used include, for example, liposomes, microcapsules, erythrocytes and the like.
  • the oligonucleotides also can be applied locally by direct injection, can be released from devices such as implanted stents or catheters, or delivered directly to the site by an infusion pump.
  • the methods of the present invention are useful in inhibiting the expression of protein encoding genes, as well as regulating non-encoding DNA such as regulatory sequences.
  • the antisense oligonucleotides are delivered to a specific defined locus, they can be used in vivo when systemic administration is not possible.
  • systemically administered oligonucleotides may be inactivated by endonucleases rendering them ineffective before they reach their targets.
  • Large doses of the oligonucleotide may be necessary for successful systemic treatment systemically, which may have harmful or toxic effects on the patient.
  • the present method provides a means for treating a large number of specific disorders using oligonucleotide therapy by delivering an antisense sequence to the specific location where it is needed.
  • Figures 1A and B are graphs of the cell count for SV-smooth muscle cells (SMC) cells treated with antisense NMMHC (A) and antisense c-myb (B) at various concentrations.
  • Figures 2A and B are graphs of the cell count for SV40TL-SMC cells, BC3H1 cells, rat aortic SMC, and mouse aortic SMC treated with antisense HNMMHC (A) and c-myb (B).
  • Figure 3A is a graph of the effect on growth of SV- SMC cells treated for different time intervals with antisense c-myb and NMMHC.
  • Figure 3B is a bar graph showing the effect on SV- SMC cells treated with unmodified antisense c-myb (light bar) and NMMHC (dark bar) for 16 hours and 40 hours after release from growth arrest.
  • Figure 4 is a bar graph of the results of a c-myb RNA dot blot, showing the amount of mRNA present in SV- SMC cells treated with sense c-myb (S Myb), antisense c-myb (AS Myb) and heparin compared with untreated (control) and growth-arrested (GA) cells.
  • Figure 5 is a graph showing the release kinetics of oligonucleotides from a PluronicTM 127 gel matrix.
  • Figure 6 is a graph showing the release kinetics of oligonucleotides from an EVAc matrix.
  • Figure 7 is a bar graph of the effect on rat arteries of antisense c-myb (AS Myb) using a PluronicTM gel and ethylene vinyl acetate matrix (EVAc) as the delivery systems for application of the oligonucleotides to the injured artery, versus intima/media ratio, a measure of neo-intimal proliferation.
  • AS Myb antisense c-myb
  • EVAc ethylene vinyl acetate matrix
  • Figure 8 is a bar graph of the effects on rat arteries of antisense c-myb applied using a PluronicTM gel compared to a drug-free gel, a gel containing sense c-myb and an untreated artery.
  • Figure 9 is a bar graph showing the effect on rabbit arteries of a mixture of antisense c-myb and human NMMHC (200 ⁇ M each) on the proliferation of cells in the artery after balloon angioplasty.
  • a method for inhibiting expression of protein- encoding genes using antisense oligonucleotides is described. The method is based on the localized application of the oligonucleotides to a specific site in vivo.
  • the oligonucleotides preferably are applied directly to the target tissue in mixture with an implant or gel, or by direct injection or infusion.
  • the oligonucleotides are treated to render them resistant in vivo to degradation or alteration by endogenous enzymes.
  • the therapeutic approach using antisense oligonucleotides is based on the principle that the function of a gene can be disrupted by preventing transcription of the gene or translation of the protein encoded by that gene. This can be accomplished by providing an appropriate length oligonucleotide which is complementary to at least a portion of the messenger RNA (mRNA) transcribed from the gene.
  • mRNA messenger RNA
  • the antisense strand hybridizes with the mRNA and targets the mRNA for destruction thereby preventing ribosomal translation, and subsequent protein synthesis.
  • the specificity of antisense oligonucleotides arises from the formation of Watson-Crick base pairing between the heterocyclic bases of the oligonucleotide and complementary bases on the target nucleic acid.
  • a nucleotide sequence sixteen nucleotides in length will be expected to occur randomly at about every 4 16 , or 4 x 10 9 nucleotides. Accordingly, such a sequence is expected to occur only once in the human genome.
  • a nucleotide sequence of ten nucleotides in length would occur randomly at about every 4 10 or lxlO 6 nucleotides.
  • Such a sequence might be present thousands of times in the human genome. Consequently, oligonucleotides of greater length are more specific than oligonucleotides of lesser length and are less likely to induce toxic complications that might result from unwanted hybridization. Therefore, oligonucleotides of the present invention are preferably at least 14 nucleotide bases in length. Oligonucleotides having from about 14 to about 38 bases are preferred, most preferably from about 15 to 30 bases.
  • the oligonucleotide sequence is selected based on analysis of the sequence of the gene to be inhibited.
  • the gene sequence can be determined, for example, by isolation and sequencing, or if known, through the literature.
  • the sequence of the oligonucleotide is an "antisense" sequence, that is, having a sequence complementary.to the coding strand of the molecule.
  • the sequence of the oligonucleotide is substantially identical to at least a portion of the gene sequence, and is complementary to the mRNA sequence transcribed from the gene.
  • the oligonucleotide therapy can be used to inhibit expression of genes from viruses or other microorganisms that are essential to infection or replication, genes encoding proteins involved in a disease process, or regulatory sequences controlling the expression of proteins involved in disease or other disorder, such as an autoimmune disorder or cardiovascular disease.
  • Oligonucleotides useful in the present invention can be synthesized by any art-recognized technique for nucleic acid synthesis.
  • the oligonucleotides are preferably synthesized using an automated synthesizer such as Model 8700 automated synthesizer (Milligen- Biosearch, Burlington, MA) , as described in detail in the Examples below, or an ABI Model 380B using H-phosphonate chemistry on controlled pore glass (CPG).
  • an automated synthesizer such as Model 8700 automated synthesizer (Milligen- Biosearch, Burlington, MA) , as described in detail in the Examples below, or an ABI Model 380B using H-phosphonate chemistry on controlled pore glass (CPG).
  • CPG controlled pore glass
  • oligonucleotide is a deoxyribonucleic acid (DNA), although ribonucleic acid (RNA) sequences may also be synthesized and applied.
  • DNA deoxyribonucleic acid
  • RNA ribonucleic acid
  • the oligonucleotides useful in the invention preferably are designed to resist degradation by endogenous nucleolytic enzymes. In vivo degradation of oligonucleotides produces oligonucleotide breakdown products of reduced length. Such breakdown products are more likely to engage in non-specific hybridization and are less likely to be effective, relative to their full-length counterparts. Thus, it is desirable to use oligonucleotides that are resistant to degradation in the body and which are able to reach the targeted cells.
  • the present oligonucleotides can be rendered more resistant to degradation in vivo by substituting one or more internal artificial internucleotide linkages for the native phosphodiester linkages, for example, by replacing phosphate with sulfur in the linkage.
  • linkages examples include phosphorothioates, methylphosphonates, sulfone, sulfate, ketyl, phosphorodithioates, various phosphoramidates, phosphate esters, bridged phyosphorothioates and bridged phosphoramidates.
  • Such examples are illustrative, rather than limiting, since other internucleotide linkages are known in the art. See, e.g., Cohen, Trends in Biotechnology (1990).
  • Oligonucleotides having one or more of these linkages substituted for the phosphodiester internucleotide linkages is well known in the art, including synthetic pathways for producing oligonucleotides having mixed internucleotide linkages.
  • Oligonucleotides can be made resistant to extension by endogenous enzymes by "capping" or incorporating similar groups on the 5' or 3' terminal nucleotides.
  • a reagent for capping is commercially available as Amino-Link IITM from Applied BioSystems, Inc., Foster City, CA. Methods for capping are described, for example, by Shaw et al..
  • the concentration of the oligonucleotides at the desired locus is much higher than if the oligonucleotides were administered systemically, and the therapeutic effect can be achieved using a significantly lower total amount.
  • the local high concentration of oligonucleotides enhances penetration of the targeted cells and effectively blocks translation of the target nucleic acid sequences.
  • the oligonucleotides can be delivered to the locus by any means appropriate for localized administration of a drug.
  • a solution of the oligonucleotides can be injected directly to the site or can be delivered by infusion using an infusion pump.
  • the oligonucleotides also can be incorporated into an implantable device which when placed at the desired site, permits the oligonucleotides to be released into the surrounding locus.
  • the oligonucleotides are most preferably administered via a hydrogel material.
  • the hydrogel is noninflammatory and biodegradeable. Many such materials now are known, including those made from natural and synthetic polymers.
  • the method exploits a hydrogel which is liquid below body temperature but gels to form a shape-retaining semisolid hydrogel at or near body temperature.
  • Preferred hydrogel are polymers of ethylene oxide-propylene oxide repeating units. The properties of the polymer are dependent on the molecular weight of the polymer and the relative percentage of polyethylene oxide and polypropylene oxide in the polymer.
  • Preferred hydrogels contain from about 10 to about 80% by weight ethylene oxide and from about 20 to about 90% by weight propylene oxide.
  • a particularly preferred hydrogel contains about 70% polyethylene oxide and 30% polypropylene oxide.
  • Hydrogels which can be used are available, for example, from BASF Corp., Parsippany, NJ, under the tradename Pluronic R .
  • the hydrogel is cooled to a liquid state and the oligonucleotides are admixed into the liquid to a concentration of about 1 mg oligonucleotide per gram of hydrogel.
  • the resulting mixture then is applied onto the surface to be treated, e.g., by spraying or painting during surgery or using a catheter or endoscopic procedures.
  • the polymer warms, it solidifies to form a gel, and the oligonucleotides diffuse out of the gel into the surrounding cells over a period of time defined by the exact composition of the gel.
  • the oligonucleotides can be administered by means of other implants that are commercially available or described in the scientific literature, including liposomes, microcapsules and implantable devices.
  • implants made of biodegradable materials such as polyanhydrides, polyorthoesters, polylactic acid and polyglycolic acid and copolymers thereof, collagen, and protein polymers, or non-biodegradable materials such as ethylenevinyl acetate (EVAc), polyvinyl acetate, ethylene vinyl alcohol, and derivatives thereof can be used to locally deliver the oligonucleotides.
  • EVAc ethylenevinyl acetate
  • the oligonucleotides can be incorporated into the material as it is polymerized or solidified, using melt or solvent evaporation techniques, or mechanically mixed with the material.
  • the oligonucleotides are mixed into or applied onto coatings for implantable devices such as dextran coated silica beads, stents, or catheters.
  • the dose of oligonucleotides is dependent on the size of the oligonucleotides and the purpose for which is it administered. In general, the range is calculated based on the surface area of tissue to be treated.
  • the effective dose of oligonucleotide is somewhat dependent on the length and chemical composition of the oligonucleotide but is generally in the range of about 30 to 3000 ⁇ g per square centimeter of tissue surface area.
  • a dose of about 320 ⁇ g oligonucleotide applied to one square centimeter of tissue was effective in suppressing expression of the c-myb gene product.
  • the oligonucleotides may be administered to the patient systemically for both therapeutic and prophylactic purposes.
  • antisense oligonucleotides specific for c-myb, NMMHC and/or PCNA may be administered to a patient who is at risk for restenosis due to angioplasty or other procedure.
  • the oligonucleotides may be administered by any effective method, for example, parenterally (e.g., intravenously, subcutaneously, intramuscularly) or by oral, nasal or other means which permit the oligonucleotides to access and circulate in the patient's bloodstream.
  • Oligonucleotides administered systemically preferably are given in addition to locally administered oligonucleotides, but also have utility in the absense of local administration.
  • a dosage in the range of from about 0.1 to about 10 grams per administration to an adult human generally will be effective for this purpose.
  • the method of the present invention can be used to treat a variety of disorders which are linked to or based on expression of a protein by a gene.
  • the method is particularly useful for treating vascular disorders, particularly vascular restenosis.
  • vascular disorders particularly vascular restenosis.
  • the following non- limiting examples demonstrate use of antisense oligonucleotides to prevent or very significantly inhibit restenosis following vascular injury such as is induced by balloon angioplasty procedures. This has been accomplished by using antisense, delivered locally, to inhibit expression of genes encoding proteins determined to be involved in vascular restenosis, including c-myb, non-muscle myosin heavy chain (NMMHC) and proliferative cellular nuclear antigen (PNAC).
  • NMMHC non-muscle myosin heavy chain
  • PNAC proliferative cellular nuclear antigen
  • the methods of the invention have many other uses.
  • oligonucleotides having a nucleotide sequence complementary to the mRNA transcript of the target gene appear to be critically involved in the initiation of proliferation of smooth muscle cells.
  • antisense oligonucleotides offers a means for treating post-angioplasty restenosis and chronic processes such as atherosclerosis, hypertension. primary pulmonary hypertension, and proliferative glomerulonephritis, which involve proliferation of smooth muscle cells.
  • pulmonary disorders such as acute respiratory distress syndrome, idiopathic pulmonary fibrosis, emphysema, and primary pulmonary hypertension. These conditions may be treated, for example, by locally delivering appropriate antisense incorporated in an aerosol by inhaler. These disorders are induced by a complex overlapping series of pathologic events which take place in the alveolus (air side), the underlying basement membrane and smooth muscle cells, and the adjacent endothelial cell surface (blood side) . It is thought that the alveolar macrophage recognizes specific antigens via the T cell receptor, become activated and elaborates a variety of substances such as PDGF which recruit white blood cells as well as stimulate fibroblasts.
  • the molecular events outlined above also induce activation of the microvascular endothelial cell surface with the appearance of selectins and integrins as well as the appearance of tissue factor which initiates blood coagulation.
  • selectin and integrin surface receptors allow white blood cells to adhere to microvascular endothelial cells and release proteases as well as other molecules which damage these cells and allow fluid to accumulate within the alveolus.
  • the above events also trigger microvascular thrombosis with closure of blood vessels. The end result of this process is to further impede oxygen exchange.
  • Antisense oligonucleotides locally delivered to the alveolar/microvascular area, could be directed against the following targets to intervene in the pathology outlined above, since the cDNA sequences of all of the targets selected are known.
  • antisense oligonucleotides specific for mRNA transcribed from the genes would inhibit production of the alveolar macrophage T cell receptor to prevent initiation of the above events; inhibit product of a protein to prevent activation of alveolar white cells, or inhibit production of elastase to prevent destruction of alveolar membrane; inhibit production of PDGF to prevent recruitment of white cells or resultant fibrosis; inhibit production of c-myb to suppress SMC proliferation; inhibit production of p-selectin or e-selectin or various integrins to prevent adhesion of blood white cells to pulmonary microvascular endothelial cells; or inhibit the production of tissue factor and PAI-1 to suppress microvascular thrombosis.
  • Tissue Factor is required for coagulation system activation.
  • Local application of antisense targeting the mRNA or DNA of a segment of TF in the area of clot formation can prevent additional coagulation.
  • This therapy can be employed as an adjunct to or as a substitute for systemic anticoagulant therapy or after fibrinolytic therapy, thereby avoiding systemic side effects.
  • Plasminogen activator inhibitor (PAI-1) is known to reduce the local level of tissue plasminogen activator (TPA) .
  • TPA tissue plasminogen activator
  • the human cDNA sequence for PAI-1 is known.
  • Local application of antisense targeting the mRNA or DNA of PAI-1 should permit a buildup of TPA in the targeted area. This may result in sufficient TPA production to naturally lyse the clot without systemic side effects. '
  • a combination of antisense-TF and antisense-PAI-1 may be utilized to maximize the efficacy of treatment of several disorders, including local post thrombolytic therapy and preventative post-angioplasty treatment.
  • Many other vascular diseases can be treated in a manner similar to that described above by identifying the target DNA or mRNA sequence.
  • the treatment of diseases which could benefit using antisense therapy include, for example, myocardial infarction, peripheral muscular disease and peripheral angioplasty, thrombophlebitis, cerebro-vascular disease (e.g., stroke, embolism), vasculitis (e.g., temporal ateritis) angina and Budd-Chiari Syndrome.
  • DNA or mRNA encoding the following proteins could be used as target sequences: growth factors and receptors, including: PDGF-AA, PDGF-AB, PDGF-BB, PDGF-alpha Receptor, PDGF-beta Receptor, k-FGF(hst), int-2, bFGF, bFGF Receptor, aFGF, aFGF Receptor, TGF-beta family (TGF beta 1,2,3, and others), TGF-beta Receptors (type I and type II), EGF, EGF Receptor (erbB-2, her, neu), erbB3, Amphiregulin, Amphiregulin receptor, Heparin binding growth factor (HBGF), HBGF Receptor, Thrombin, Thrombin Receptor, Serum-derived growth factor (SDGF), SDGF Receptor, Interleukins (IL-1,2,3,4,5
  • A, B, embryonic, etc. Myosin non-regulatory light chain, Myosin essential light chain, Myosin light chain kinase, Myosin phophatase, cAMP kinase, cAMP phosphatase, Myosin heavy chain kinase, 62 kDa Ca-binding protein, Calpain II, Dynein, Kinesin, INCENP proteins; signal transduction factors, including: src, MAP4, MAP Kinase, GMRF, GAP, NF-1, NF-kappa B, PI-3K, PLC-gamma, SRF, yes, fps (fes), abl, met, mos, raf (mil), Ha-ras, Ki-ras, N-ras, crk; adhesion proteins, including: ICAM-1, VCAM, LCAM, Integrin Family, Tensin, FAK, Thrombomodulin; and calcium
  • SV40LT-SMC rat smooth muscle cells, gift of Dr. C. Reilly, Merck, Sharp and Dohme, West Point, PA
  • DMEM Dulbecco's modified Eagle's Medium
  • FBS heat-inactivated fetal bovine serum
  • SMC Primary aortic smooth muscle cells
  • Antisense and sense 18-mer phosphorothiolate oligonucleotides were synthesized on an ABI DNA synthesizer. Oligonucleotides were deprotected on the machine, dried down, resuspended in "TE" (10 mM Tris, pH 7.5, lmM EDTA, pH8.0) and quantified by spectrophotometry and gel electrophoresis. The following sequences were employed: Antisense c-myb oligonucleotide:
  • Antisense NMMHC oligonucleotide
  • TM Antisense thrombomodulin
  • Antisense human c-myb oligonucleotide which has 2 mismatches compared with mouse c-myb:
  • Sequence ID No. 1 is complementary to nucleotides 4-22 of mouse c-myb (Bender et al., (1986) Natl. Acad. Sci. USA,83:3204-3208); Sequence ID No. 2 is complementary to nucleotides 232-250 of human NMMHC-A (Simons et al., (1991) Cir. Res. ,69:530-539) ; and
  • Sequence ID No. 3 is complementary to nucleotides 4-25 of mouse TM (Dittman and Majerus, (1989) Nucl. Acid Res.,17:802) .
  • Sequence ID No. 4 is complementary to a human c-myb sequence and has 2 base mismatches compared with urine c-myb.
  • the NMMHC sequence was chosen in a region with the closest degree of homology between known nonmuscle myosin sequences.
  • the sequence has 1 nucleotide difference with a human NMMHC-B (Simons et al., ibid.) and 2 nucleotide differences with chicken NMMHC-A and NMMHC-B. The corresponding sense sequences were used as controls.
  • Example 1 Inhibition of c-myb and NMMHC-A using antisense oligonucleotides in vitro.
  • Both cell lines as well as early passage primary aortic SMC were seeded at a density of 25,000 cells per well in cluster 6 well plates (Costar, Cambridge, MA) in 10% FBS-DMEM (20% FBS-DMEM for BC3H1 cells). The following day, the cells were washed twice with phosphate-buffered saline (PBS) , the media was replaced with 0.5% FBS-DMEM growth arrest media, and the cells were kept in growth-arrest media for 96 hours. The media then was changed to 10% or 20% FBS-DMEM, and synthetic c-myb and NMMHC antisense and sense oligonucleotides were added. The cells were permitted to grow for 72 hours, trypsinized and counted on a Coulter Counter.
  • PBS phosphate-buffered saline
  • the two cell lines and the SMC cells were allowed to proliferate in 10% or 20% FBS-DMEM, oligonucleotides were added, and cell counts were obtained after 5-8 days as described above. Each experiment was carried out in triplicate and repeated at least two additional times. Data is expressed as mean + standard deviation * The results (shown in Figure 1) showed that, in vitro, antisense oligonucleotides to both c-myb (Seq. ID No. 1) and NMMHC (Seq. ID No. 2) caused substantial suppression of cellular proliferation while the sense oligonucleotides had no effect and were similar to the results obtained using just Tris-EDTA buffer.
  • the oligonucleotides utilized were derived from the nucleotide sequences of human/chicken NMMHC or mouse c-myb cDNAs. The importance of the specificity of the antisense oligonucleotides was shown by the complete loss of antiproliferative action when two bases of the 18 base c-myb antisense sequence were randomly altered (Seq. ID No. 4). The results for this test were as follows: antisense c-myb: 475,600 cells + 25,000 cells; mismatch antisense c-myb: 958,800 cells + 12,000 cells; sense c-myb: 935,200 cells + 22,000 cells. Thus, the mismatch antisense c-myb (seq. ID No.
  • TM phosphorothiolate thrombomodulin
  • antisense phosphorothiolate oligonucleotides directed against NMMHC as compared to c-myb was more clearly concentration-dependent (antisense NMMHC: 32% vs 65% suppression at 2 ⁇ M and 25 ⁇ M, respectively; antisense c-myb: 33% vs 50% suppression at 2 ⁇ M and 25 ⁇ M respectively).
  • antisense NMMHC 32% vs 65% suppression at 2 ⁇ M and 25 ⁇ M, respectively
  • antisense c-myb 33% vs 50% suppression at 2 ⁇ M and 25 ⁇ M respectively.
  • Previous estimates of the relative abundance of these two messages indicated that c-myb mRNA occurs at extremely low concentrations in exponentially growing SMC (less than 0.01% of poly A+ RNA), whereas NMMHC mRNA is present at significantly higher levels.
  • the observed concentration dependence of the two antisense oligonucleotides with regard to growth inhibition was consistent with the relative abundance of the two mRNAs.
  • the antiproliferative effects of the antisense and sense phosphorothiolate oligonucleotides were also evaluated with the BC3H1 cell line as well as with primary rat and mouse aortic SMC. The data obtained showed that growth of the three cell types is greatly suppressed with phosphorothiolate antisense but not sense NMMHC or c-myb oligonucleotides ( Figure 2).
  • Antisense c-myb oligonucleotides exhibited a greater antiproliferative effect on mouse aortic SMCs and mouse BC3H1 cells as compared to rat aortic SMC and rat SV40LT-SMC ( Figure 2B) .
  • the difference in growth inhibition is most likely attributable to the greater extent of antisense nucleotide mismatch between rat and mouse c-myb sequences within the chosen area.
  • the minimal time required for exposure of SV40LT-SMC to antisense NMMHC or c-myb ' phosphorothiolate oligonucleotides to achieve maximal growth inhibition was determined.
  • SV40LT-SMC were allowed to grow exponentially while continuously exposed to 10 ⁇ M antisense NMMHC or c-myb oligonucleotides and cell counts were determined at 72 hr and 120 hr.
  • the treatment of SMC with antisense NMMHC oligonucleotides produced no growth inhibitory effect at either time point, whereas exposure to antisense c-myb oligonucleotides generated a 19% suppression of proliferation at 72 hr and a 40% suppression of proliferation at 120 hr.
  • RNA Analysis Total cellular RNA was determined from SV40LT-SMC cells in culture 24 hours after growth induction with 10% FBS in DMEM using the method of Chomzynski and Sacchi, J. Cell. Physiol., 1 2_:342 (1990). The RNA was quantified by spectrophotometry and a total of 10 " ⁇ g was applied to nitrocellulose using a dot blot apparatus. The blot was then hybridized with a random primed c-myb, NMMHC, large T-antigen, GAPDH and TM probes in 10% dextran sulfate and 40% formamide for 16 hours at 42°C.
  • Northern blots and RNA dot blots were washed in SSC solution with final washes performed at 50°C and 0.5 x SSC for c-myb, 55°C and 0.2 x SSC for NMMHC, 55°C and 0.2 x SSC for large T antigen, 50°C and 0.2 x SSC for GADPH and 50°C and 0.2 x SSC for TM.
  • the Northern blots were subjected to autoradiography.
  • the RNA blots were quantified by normalizing c-myb or GAPDH message counts to large T-antigen counts using a Betascope 603 analyzer (Betagen, Waltham, MA). The numbers represent total counts for each dot. The entire experiment was repeated twice.
  • Results are for cells treated with sense c-myb oligonucleotide (25 ⁇ M); antisense c-myb oligonucleotide (25 ⁇ M); and heparin (100 ⁇ g/ml). The cells were allowed to reach confluence and become quiescent for two days. Cells exposed to antisense c-myb oligonucleotide had a markedly decreased amount of c-myb message present as assessed by RNA dot blot hybridization with a radiolabeled c-myb probe. The results are shown in Figure 4.
  • Figure 4 shows individual dot counts, adjusted for the quantity of RNA in each sample, for synchronized proliferating SV-SMCs treated with sense c-myb (S Myb) and antisense c-myb (AS Myb), heparin- arrested (heparin), and growth-arrested (GA) cells.
  • Cell growth was arrested with antisense c-myb to about the same degree as with heparin had similar amounts of c-myb message present.
  • antisense NMMHC oligonucleotide led to a marked attenuation of the NMMHC message detected on a Northern blot.
  • the amount of c-myb protein in the cells treated with antisense c-myb was markedly reduced, as was the non-muscle myosin protein in cells treated with antisense NMMHC-B, as assessed by indirect immunofluorescence.
  • SV40LT-SMC were fixed with 2% formaldehyde/PBS at room temperature for 15 minutes, permeated with 2% Triton X-100/PBS, washed 3 times with 1% BSA/PBS and exposed for 2-4 hr to anti-myb or anti-NMMHC antisera diluted 1:250 or 1:1000 in 1% BSA/PBS.
  • the anti-myb antisera was obtained from Cambridge Research Laboratories (Wilmington, DE) and was generated by immunizing rabbits with the synthetic peptide His-Thr- Cys-Ser-Tyr-Pro-Gly-Trp-His-Ser-Thr-Ser-IIe-Val corresponding to mouse c-myb amino acid residues 332- 345.
  • the anti-nonmuscle myosin antiserum was kindly provided by RS Adelstein and JS Sellers (LMC, NIH, Bethesda, MD) and was generated by immunizing rabbits with purified human platelet myosin. This antiserum is monospecific as judged by Western blot analysis.
  • the cells were washed three times with 1% BSA/PBS to remove excess primary antibody followed by incubation for 2 hr with second antibodies diluted 1:100 in 1% BSA/PBS (rhodamine-conjugated goat anti-rabbit IgG and FITC- conjugated sheet anti-rabbit purchased from Organon Teknika, Durham, NC). After washing cells three times with 1% BSA/PBS, the samples were examined with a Nikon Optiphot fluorescence photomicroscope.
  • SV40LT-SMC cells were plated at low density (10,000/cm 2 ) on a 2 well glass slides (Nunc, Inc., Naperville, IL), growth-arrested for 96 hr in 0.5% FBS-DMEM and then shifted to 10% FBS- DMEM to which were added 25 ⁇ M antisense or sense NMMHC or c-myb phosphorothiolate oligonucleotides.
  • Example 2 Release of oligonucleotides from polymeric matrices.
  • the release kinetics of the gels containing oligonucleotides were determined by placing the gels in PBS and measuring the absorption (OD) over time. The results for four test gels, shown in Figure 5, indicate that oligonucleotides are released from the gels in less than one hour. Release of oligonucleotides from EVAc matrices The release of oligonucleotides from ethylene vinyl acetate (EVAc) matrices was demonstrated. Matrices were constructed and release was determined as described by Murray et al. (1983), In Vitro., 1 ⁇ :743-748.
  • Ethylene-vinyl acetate (EVAc) copolymer (ELVAX 40P, DuPont Chemicals, Wilmington, DE) was dissolved in dichloromethane to form a 10% weight by volume solution.
  • Bovine serum albumin and the oligonucleotide were dissolved together at a ratio of 1000 - 2000:1 in deionized HO, frozen with liquid N and then lyophilized to form a dry powder.
  • the powder was pulverized to form a homogeneous distribution of particles less than 400 microns in diameter.
  • a known quantity of the powder was combined with 4-10 ml of the 10% (w/v) EVAc copolymer solution in a 22 ml glass scintillation vial.
  • the vial was vortexed for 10 seconds to form a homogeneous suspension of the drug particles in the polymer solution.
  • This suspension was poured onto a glass mold which had been precooled on a slab of dry ice. After the mixture froze it was left in place for 10 minutes and then removed from the mold and placed into a -20°C freezer for 2 days on a wire screen.
  • the slab was dried for an additional 2 days at 23°C under a 600 millitorr vacuum to remove residual dichloromethane. After the drying was complete 5 mm X 0.8 mm circular slabs are excised with a #3 cork borer. The results, shown in Figure 6, indicate that about 34% of the oligonucleotide was released within the first 48 hours.
  • Example 3 In vivo application of oligonucleotides to inhibit c-myb and NMMHC in rats. I. Animal Model.
  • Balloon stripping of the rat carotid artery is used as a model of restenosis ⁇ n vivo.
  • Rats were anesthetized with Nembutal (50 mg/kg).
  • a left carotid dissection was carried out and a 2F Fogarty catheter was introduced' through the arteriotomy incision in the internal carotid artery.
  • the catheter was advanced to the aortic arch, the balloon was inflated and the catheter withdrawn back to the arteriotomy site. This was repeated two more times. Subsequently, the balloon being withdrawn, the internal carotid was tied off, hemostasis achieved, and the wound closed.
  • the oligonucleotides were applied with a hydrogel and with an implantable ethylene vinyl acetate (EVAc) matrix.
  • EVAc implantable ethylene vinyl acetate
  • a polyethylene oxide-polypropylene oxide polymer (PluronicTM 127, BASF, Parsippany, NJ) was used as a hydrogel.
  • the PluronicTM gel matrices were prepared as described in Example 2. Briefly, sterile solutions of PluronicTM 127 were prepared by weighing 1.25 g of UV sterilized Pluronic powder into a scintillation vial and adding 3.25 ml of sterile water. Solubilization was achieved by cooling on ice while shaking, forming a solution containing 27.7% by weight of the polymer.
  • oligonucleotides 5.041 mg/500 ⁇ L
  • the final gels were 25% w/w of PluronicTM polymer and lmg/g oligonucleotide.
  • Drug-free 25% (w/w) gels were prepared as controls.
  • the EVAc matrices were prepared as described in Example 2, and contained 40 ⁇ g of oligonucleotide.
  • 200 ⁇ l of Pluronic/oligonucleotide solution which contained 200 ⁇ g of the oligonucleotide
  • the antisense/EVAc matrix which contained 40 ⁇ g of the oligonucleotide
  • drug-free gels were applied in the same manner.
  • Figure 8 shows the results of extension of this experiment, in which 28 rats were treated as described above. Seven rats in each treatment group were subjected to balloon angioplasty, and the arterial walls treated as follows: with a drug free hydrogel (PluronicTM127 as described above), a hydrogel containing sense c-myb, a hydrogel containing antisense c-myb, and no treatment at all. As shown in Figure 8, similar high levels of neointimal proliferation occurred in all animals except those treated with antisense c-myb, where the levels of proliferation were dramatically lower.
  • a drug free hydrogel PluronicTM127 as described above
  • Example 4 Inhibition of PCNA using antisense oligonucleotides.
  • Sense PCNA was used as a negative control
  • NMMHC-B was used as a positive (inhibitory) control.
  • Example 5 In vivo application of antisense oligonucleotides to inhibit smooth muscle cell proliferation in rabbits.
  • New Zealand white rabbits (1-1.5 Kg) were anesthetized with a mixture of ketamine and zylazine and carotid dissection was performed as described in Example 3.
  • a 5F Swan-Ganz catheter was inserted and positioned in the descending aorta with fluoroscopic guidance.
  • the Swan-Ganz catheter was exchanged over the wire for an angioplasty catheter with a 3.0 mm balloon.
  • the common iliac artery was angioplastied 3 times at 100 PSI for 90 seconds each time.
  • a Wolinsky catheter was introduced and loaded with oligonucleotide solution in a total volume of 5cc normal saline. Saline was injected as a control in a counterlateral iliac artery.
  • the oligonucleotides were a mixture of antisense mouse c-myb and human NMMHC (200 ⁇ M of each), described above. The mixture was injected under 5 atmospheres of pressure over 60 seconds. Two rabbits were treated with antisense oligonucleotide.
  • the animals were sacrificed 4 weeks later and the arteries were processed as described in Example 3 for rat arteries.
  • Example 6 Inhibition of proliferation of baboon smooth muscle cells using antisense oligonucleotides.
  • primary baboon smooth muscle cells gifts from Dr. Hawker, Emory University
  • human NMMHC Seq. ID No. 2
  • the cells were allowed to grow for 72 hours after treatment with the oligonucleotides, then counted as described in Example 1.
  • the results show that hNMMHC caused 65.5% growth suppression and c-myb caused 59.77% growth suppression in the baboon cells.
  • ROSENBERG ROBERT D. SIMONS, MICHAEL EDELMAN, ELAZER
  • ADDRESSEE TESTA, HURWITZ & THIBEAULT
  • B STREET: 53 STATE STREET
  • MOLECULE TYPE DNA (synthetic)
  • HYPOTHETICAL NO
  • ANTI-SENSE YES
  • MOLECULE TYPE DNA (synthetic)
  • HYPOTHETICAL NO
  • ANTI-SENSE YES
  • FEATURE FEATURE:
  • MOLECULE TYPE DNA (synthetic)
  • HYPOTHETICAL NO
  • ANTI-SENSE YES
  • FEATURE FEATURE:
  • MOLECULE TYPE DNA (synthetic)
  • HYPOTHETICAL NO

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Abstract

Procédé d'application localisée d'oligonucléotides anti-sens. Le procédé s'est avéré être efficace pour inhiber l'expression et la traduction de divers gènes. Le procédé emploie des oligonucléotides anti-sens spécifiques de l'ARN messager transcrit à partir du gène à examiner. On applique directement sur le locus voulu des cellules à traiter lesdits oligonucléotides anti-sens, et ces derniers s'hybrident avec ledit ARN messager et inhibent l'expression du gène. On a également prévu des dispositifs d'application localisée d'anti-sens ainsi que leurs procédés de fabrication.
PCT/US1992/009626 1991-11-08 1992-11-05 Traitement par application localisee d'oligonucleotides WO1993008845A1 (fr)

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Cited By (21)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996005321A1 (fr) * 1994-08-17 1996-02-22 Rhone-Poulenc Rorer S.A. Therapie genique de la restenose au moyen de vecteur adenovial
EP0723440A1 (fr) * 1993-10-15 1996-07-31 Cytrx Corporation Compositions d'apport therapeutique et leurs modes d'utilisation
EP0750503A1 (fr) * 1994-03-07 1997-01-02 Immusol, Inc. Therapie a base de ribozymes pour traiter la restenose
US5646042A (en) * 1992-08-26 1997-07-08 Ribozyme Pharmaceuticals, Inc. C-myb targeted ribozymes
US5658780A (en) * 1992-12-07 1997-08-19 Ribozyme Pharmaceuticals, Inc. Rel a targeted ribozymes
EP0789564A1 (fr) * 1994-11-18 1997-08-20 Supratek Pharma, Inc. Composes polynucleotidiques
EP0871496A1 (fr) * 1995-04-19 1998-10-21 Thomas Jefferson University Procedes et compositions pour les traitements de greffes arterio-veineuses et veineuses
JP2000506165A (ja) * 1996-03-04 2000-05-23 ザ ペン ステイト リサーチ ファウンデーション 細胞インターナリゼーションを増強するための物質および方法
US6103705A (en) * 1996-11-27 2000-08-15 Aventis Pharmaceuticals Products Inc. Pharmaceutical composition comprising a compound having anti-Xa activity and a platelet aggregation antagonist compound
US6133242A (en) * 1993-10-15 2000-10-17 Thomas Jefferson Univerisity Inhibition of extracellular matrix synthesis by antisense compounds directed to nuclear proto-oncogenes
US6159946A (en) * 1993-01-07 2000-12-12 Thomas Jefferson University Antisense inhibition of c-myc to modulate the proliferation of smooth muscle cells
WO2001074900A2 (fr) 2000-03-31 2001-10-11 Aventis Pharmaceuticals Products Inc. Facteur inducteur du facteur nucleaire kb
US6933286B2 (en) 1991-03-19 2005-08-23 R. Martin Emanuele Therapeutic delivery compositions and methods of use thereof
US7202225B1 (en) 1993-10-15 2007-04-10 Emanuele R Martin Therapeutic delivery compositions and methods of use thereof
EP2060630A2 (fr) 1997-04-10 2009-05-20 Stichting Katholieke Universiteit University Medical Centre Nijmegen PCA3, gènes de PCA3, et procédés d'utilisation
US7655408B2 (en) 1999-09-29 2010-02-02 Diagnocure Inc. PCA3 messenger RNA species in benign and malignant prostate tissues
USRE41996E1 (en) 1996-03-04 2010-12-14 The Penn State Research Foundation Massachusetts Institute of Technology Composition and methods for enhancing receptor-mediated cellular internalization
US7960109B2 (en) 2004-12-24 2011-06-14 Stichting Katholieke Universiteit, The University Medical Centre Nijmegen mRNA ratios in urinary sediments and/or urine as a prognostic and/or theranostic marker for prostate cancer
US8192931B2 (en) 2003-02-07 2012-06-05 Diagnocure Inc. Method to detect prostate cancer in a sample
US8329669B2 (en) 2006-07-28 2012-12-11 Sanofi Composition and method for treatment of tumors
US8623831B2 (en) 2000-03-31 2014-01-07 Aventis Pharmaceuticals Inc. Nuclear factor κB inducing factor

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4740463A (en) * 1984-04-13 1988-04-26 Massachusetts Institute Of Technology Methods and artificial genes for antagonizing the function of an oncogene
US4806463A (en) * 1986-05-23 1989-02-21 Worcester Foundation For Experimental Biology Inhibition of HTLV-III by exogenous oligonucleotides
WO1991003260A1 (fr) * 1989-09-01 1991-03-21 Temple University Of The Commonwealth System Of Higher Education Oligonucleotides non codants pour le proto-oncogene c-abl
WO1991012811A1 (fr) * 1990-02-26 1991-09-05 Isis Pharmaceuticals, Inc. Therapies utilisant des oligonucleotides pour moduler les effets d'herpesvirus
WO1991015226A1 (fr) * 1990-04-09 1991-10-17 The American National Red Cross Compositions de rajeunissement et procedes d'utilisation

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4740463A (en) * 1984-04-13 1988-04-26 Massachusetts Institute Of Technology Methods and artificial genes for antagonizing the function of an oncogene
US4806463A (en) * 1986-05-23 1989-02-21 Worcester Foundation For Experimental Biology Inhibition of HTLV-III by exogenous oligonucleotides
WO1991003260A1 (fr) * 1989-09-01 1991-03-21 Temple University Of The Commonwealth System Of Higher Education Oligonucleotides non codants pour le proto-oncogene c-abl
WO1991012811A1 (fr) * 1990-02-26 1991-09-05 Isis Pharmaceuticals, Inc. Therapies utilisant des oligonucleotides pour moduler les effets d'herpesvirus
WO1991015226A1 (fr) * 1990-04-09 1991-10-17 The American National Red Cross Compositions de rajeunissement et procedes d'utilisation

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US6933286B2 (en) 1991-03-19 2005-08-23 R. Martin Emanuele Therapeutic delivery compositions and methods of use thereof
US5817796A (en) * 1992-08-26 1998-10-06 Stinchcomb; Dan T. C-myb ribozymes having 2'-5'-linked adenylate residues
US5646042A (en) * 1992-08-26 1997-07-08 Ribozyme Pharmaceuticals, Inc. C-myb targeted ribozymes
US5658780A (en) * 1992-12-07 1997-08-19 Ribozyme Pharmaceuticals, Inc. Rel a targeted ribozymes
US6410224B1 (en) 1992-12-07 2002-06-25 Ribozyme Pharmaceuticals, Inc. Ribozyme treatment of diseases or conditions related to levels of NF-κB
US6159946A (en) * 1993-01-07 2000-12-12 Thomas Jefferson University Antisense inhibition of c-myc to modulate the proliferation of smooth muscle cells
US6323184B1 (en) 1993-10-15 2001-11-27 Thomas Jefferson University Arteriovenous and venous graft treatments: methods and compositions
US6133242A (en) * 1993-10-15 2000-10-17 Thomas Jefferson Univerisity Inhibition of extracellular matrix synthesis by antisense compounds directed to nuclear proto-oncogenes
EP0723440A4 (fr) * 1993-10-15 1997-06-11 Cytrx Corp Compositions d'apport therapeutique et leurs modes d'utilisation
US7202225B1 (en) 1993-10-15 2007-04-10 Emanuele R Martin Therapeutic delivery compositions and methods of use thereof
EP0723440A1 (fr) * 1993-10-15 1996-07-31 Cytrx Corporation Compositions d'apport therapeutique et leurs modes d'utilisation
EP0750503A4 (fr) * 1994-03-07 1999-07-21 Immusol Inc Therapie a base de ribozymes pour traiter la restenose
EP0750503A1 (fr) * 1994-03-07 1997-01-02 Immusol, Inc. Therapie a base de ribozymes pour traiter la restenose
FR2723697A1 (fr) * 1994-08-17 1996-02-23 Rhone Poulenc Rorer Sa Methode de traitement de la restenose par la therapie genique
WO1996005321A1 (fr) * 1994-08-17 1996-02-22 Rhone-Poulenc Rorer S.A. Therapie genique de la restenose au moyen de vecteur adenovial
EP0789564A1 (fr) * 1994-11-18 1997-08-20 Supratek Pharma, Inc. Composes polynucleotidiques
EP0789564A4 (fr) * 1994-11-18 2000-11-08 Supratek Pharma Inc Composes polynucleotidiques
EP0871496A1 (fr) * 1995-04-19 1998-10-21 Thomas Jefferson University Procedes et compositions pour les traitements de greffes arterio-veineuses et veineuses
EP0871496A4 (fr) * 1995-04-19 1998-10-21
USRE42012E1 (en) 1996-03-04 2010-12-28 The Penn State Research Foundation Compositions and methods for enhancing receptor-mediated cellular internalization
EP0885002B1 (fr) * 1996-03-04 2011-05-11 The Penn State Research Foundation Materiaux et procedes permettant d'accroitre la penetration intracellulaire
JP2000506165A (ja) * 1996-03-04 2000-05-23 ザ ペン ステイト リサーチ ファウンデーション 細胞インターナリゼーションを増強するための物質および方法
USRE42072E1 (en) 1996-03-04 2011-01-25 The Penn State Research Foundation Compositions and methods for enhancing receptor-mediated cellular internalization
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US6103705A (en) * 1996-11-27 2000-08-15 Aventis Pharmaceuticals Products Inc. Pharmaceutical composition comprising a compound having anti-Xa activity and a platelet aggregation antagonist compound
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US9540696B2 (en) 1997-04-10 2017-01-10 Stichting Katholieke Universiteit, The University Medical Centre Nijmegen PCA3 genes
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US7655408B2 (en) 1999-09-29 2010-02-02 Diagnocure Inc. PCA3 messenger RNA species in benign and malignant prostate tissues
US8241848B2 (en) 1999-09-29 2012-08-14 Diagnocure Inc. Distinguishing PCA3 messenger RNA species in benign and malignant prostate tissues
US8618276B2 (en) 1999-09-29 2013-12-31 Diagnocure Inc. Distinguishing PCA3 messenger RNA species in benign and malignant prostate tissues
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US8642739B2 (en) 2000-03-31 2014-02-04 Aventis Pharmaceuticals Inc. Nuclear factor κB inducing factor
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US7960109B2 (en) 2004-12-24 2011-06-14 Stichting Katholieke Universiteit, The University Medical Centre Nijmegen mRNA ratios in urinary sediments and/or urine as a prognostic and/or theranostic marker for prostate cancer
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