WO2000009672A9 - Dnazymes et methodes de traitement de la restenose - Google Patents

Dnazymes et methodes de traitement de la restenose

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Publication number
WO2000009672A9
WO2000009672A9 PCT/IB1999/001484 IB9901484W WO0009672A9 WO 2000009672 A9 WO2000009672 A9 WO 2000009672A9 IB 9901484 W IB9901484 W IB 9901484W WO 0009672 A9 WO0009672 A9 WO 0009672A9
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WIPO (PCT)
Prior art keywords
dnazyme
restenosis
onset
myc
inhibiting
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Ceased
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PCT/IB1999/001484
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English (en)
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WO2000009672A1 (fr
Inventor
Lun-Quan Sun
Murray J Cairns
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Johnson and Johnson Research Pty Ltd
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Johnson and Johnson Research Pty Ltd
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Priority to KR1020017001893A priority Critical patent/KR20010072475A/ko
Priority to JP2000565109A priority patent/JP2002525037A/ja
Priority to AU52984/99A priority patent/AU5298499A/en
Priority to CA002340322A priority patent/CA2340322A1/fr
Priority to EP99938479A priority patent/EP1117768A4/fr
Publication of WO2000009672A1 publication Critical patent/WO2000009672A1/fr
Publication of WO2000009672A9 publication Critical patent/WO2000009672A9/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system

Definitions

  • This invention relates to inhibiting the onset of restenosis using DNAzymes.
  • the DNAzymes accomplish this end by cleaving mRNA encoding c-myc, whose expression in vascular smooth muscle cells is required" for restenosis to occur.
  • Restenosis is a serious medical disorder which often occurs following angioplasty. This disorder afflicts 30%-60% of all angioplasty patients.
  • Restenosis is understood to be caused, at least in part, by excessive proliferation of smooth muscle cells
  • SMC's following vascular injury occurring during angioplasty.
  • Several biological modulators are thought to facilitate this SMC proliferation. These modulators include platelet-derived growth factor (“PDGF”) , fibroblast growth factor (“FGF”) and insulin growth factor (“IGF”) (Ross; Banscota; Libby; Gay) .
  • PDGF platelet-derived growth factor
  • FGF fibroblast growth factor
  • IGF insulin growth factor
  • the induction of SMC proliferation by these modulators occurs via the intracellular transactivation of a number of important genes (Kindy; Gadeau) . These genes include c-myc, c-my£>, c-jfos and PCNA (proliferating cell nuclear antigen) , and generally are cell cycle- specific.
  • the c-myc gene is over-expressed in SMC's within 30 minutes to two hours of vascular trauma, and expression declines to normal levels within 12 hours thereafter.
  • angioplasty causes vascular SMC injury, which triggers excess c-myc expression beginning 30 minutes to two hours after injury, and ending 12 hours after injury.
  • Radioactive implants include either radioactive implants or delivery of a radioactive composition to the site being treated. Although radiation therapy has shown some promising results, the long-term side effects of intra-coronary radiation have yet to be established. Regarding pharmacological therapy, both the anti-thrombotin and anti-proliferation approaches employed to date are generally ineffective (Bennet) .
  • antisense nucleic acid technology has been one of the major tools of choice to inactivate genes whose expression causes disease and is thus undesirable.
  • the anti-sense approach employs a nucleic acid molecule that is complementary to, and thereby hybridizes with, an mRNA molecule encoding an undesirable gene. Such hybridization leads to the inhibition of gene expression.
  • Anti-sense technology suffers from certain drawbacks.
  • Anti-sense hybridization results in the formation of a DNA/target mRNA heteroduplex.
  • This heteroduplex serves as a substrate for RNAse H-mediated degradation of the target mRNA component.
  • the DNA anti-sense molecule serves in a passive manner, in that it merely facilitates the required cleavage by endogenous RNAse H enzyme.
  • This dependence on RNAse H confers limitations on the design of anti-sense molecules regarding their chemistry and ability to form stable heteroduplexes with their target mRNA' s .
  • Anti- sense DNA molecules also suffer from problems associated with non-specific activity and, at higher concentrations, even toxicity.
  • catalytic nucleic acid molecules As an alternative to anti-sense molecules, catalytic nucleic acid molecules have shown promise as- therapeutic agents for suppressing gene expression, and are widely discussed in the literature (Haseloff; Breaker (1994) ; Koizumi; Otsuka; Kashani-Sabet; Raillard; and Carmi) .
  • a catalytic nucleic acid molecule functions by actually cleaving its target mRNA molecule instead of merely binding to it.
  • Catalytic nucleic acid molecules can only cleave a target nucleic acid sequence if that target sequence meets certain minimum requirements.
  • the target sequence must be complementary to the hybridizing regions of the catalytic nucleic acid, and the target must contain a specific sequence at the site of cleavage.
  • RNA molecules Catalytic RNA molecules (“ribozymes”) are well documented (Haseloff; Symonds; and Sun), and have been shown to be capable of cleaving both RNA
  • Ribozymes are highly susceptible to enzymatic hydrolysis within the cells where they are intended to perform their function. This in turn limits their pharmaceutical applications.
  • DNAzymes are single-stranded, and cleave both RNA (Breaker (1994); Santoro) and DNA (Carmi) .
  • a general model for the DNAzyme has been proposed, and is known as the "10-23" model.
  • DNAzymes following the "10-23” model also referred to simply as “10-23 DNAzymes", have a catalytic domain of 15 deoxyribonucleotides, flanked by two substrate- recognition domains of seven to nine deoxyribonucleotides each. In vitro analyses show that this type of DNAzyme can effectively cleave its substrate RNA at purine:pyrimidine junctions under physiological conditions (Santoro) .
  • DNAzymes show promise as therapeutic agents. However, DNAzyme success against a disease caused by the presence of a known mRNA molecule is not predictable. This unpredictability is due, in part, to two factors. First, certain mRNA secondary structures can impede a DNAzyme' s ability to bind to and cleave its target mRNA. Second, the uptake of a DNAzyme by cells expressing the target mRNA may not be efficient enough to permit therapeutically meaningful results. For these reasons, merely knowing of a disease and its causative target mRNA sequence does not alone allow one to reasonably predict the therapeutic success of a
  • This application provides a DNAzyme which specifically cleaves c-myc mRNA, comprising (a) a catalytic domain that has the nucleotide sequence GGCTAGCTACAACGA and cleaves mRNA at any purine :pyrimidine cleavage site at which it is directed, (b) a binding domain contiguous with the 5' end of the catalytic domain, and (c) another binding domain contiguous with the 3' end of the catalytic domain, wherein the binding domains are complementary to, and therefore hybridize with, the two regions immediately flanking the purine residue of the cleavage site within the c-myc mRNA, respectively, at which
  • each binding domain is at least six nucleotides in length, and both binding domains have a combined total length of at least 14 nucleotides.
  • This invention also provides a pharmaceutical composition for inhibiting the onset of restenosis, which comprises the instant DNAzyme and a pharmaceutically acceptable carrier suitable for topical administration.
  • This invention further provides an angioplastic stent for inhibiting the onset of restenosis, which comprises an agioplastic stent operably coated with a prophylactically effective dose of the instant pharmaceutical composition.
  • This invention still further provides a method for inhibiting the onset of restenosis in a subject undergoing angioplasty, which comprises topically administering a prophylactically effective dose of the instant pharmaceutical composition to the subject at around the time of the angioplasty.
  • this invention provides a method for inhibiting the onset of restenosis in a subject undergoing angioplasty, which comprises topically administering the instant angioplastic stent to the subject at the time of the angioplasty.
  • Figure 1 shows the structure of a "10-23" DNAzyme described in Santoro.
  • the cleavage site is indicated by an asterisk between X and Y.
  • the substrate-binding domains are indicated by N' s .
  • Figure 2 shows c-myc RNA-cleaving DNAzyme designs.
  • the cleavage site for the c-myc DNAzyme was chosen at the AUG start codon of the human c-myc mRNA (2nd exon) . Cleavage occurs between A and U as indicated.
  • Figure 3 shows the optimization of DNAzyme arm-length and chemical modification.
  • C-myc-cleaving DNAzymes with different arm lengths were designed based on the "10-23" model.
  • the 3' -3' terminal base inversion at the 3' end is indicated by a shadow C or G (3'INV) .
  • Figure 4 shows the analysis of multiple turnover kinetics.
  • Panel A shows a densitometric image, obtained using a Phosphorlmager (Molecular Dynamics) , of a 16% polyacrylamide gel, showing cleavage of synthetic c-myc mRNA under multiple turnover conditions. All reactions were performed with 200 pM DNAzyme and 2 nM, 4 nM, 8 nM, 16 nM, and 32 nM of substrate mRNA (as indicated) . The incubation time for each reaction, ranging from 0-60 minutes, is indicated at the top of each lane.
  • Panel B shows a plot of DNAzyme cleavage progress (nM) for each substrate concentration. These data were derived from densitometry measurements of cleaved bands shown in Panel A.
  • Figure 5 shows the in vitro cleavage of c-myc mRNA.
  • 1.5 kb c-myc mRNA substrate were transcribed from a pGEM vector in the presence of ⁇ 2 P-UTP.
  • the cleavage reaction was performed at 10 mM MgCl2/ 50 mM Tris.HCl, pH 7.5, 37°C for 60 minutes.
  • Figure 6 shows a stability assay of the 3 '-inverted DNAzyme in human serum.
  • DNAzymes were incubated with AB-type human serum (Sigma) . Samples were collected at different time points as indicated, and labeled with 32 P. The labeled DNAzymes were analyzed on 16% PAGE gel. Typical gel patterns are shown here for unmodified (top right) and 3' inverted DNAzymes (bottom right) .
  • Figure 7 shows the testing of c-myc mRNA-cleaving DNAzymes in SV-LT-SMC's.
  • Growth-arrested SMC's were stimulated with 10% FBS-DME (Dulbecco's Modified Eagle Medium containing 0.5% fetal bovine serum) in the presence of 10 mM anti-c-myc mRNA DNAzyme designated Rs-6 (described below) , 10 mM control oligonucleotide (same arm sequences as Rs-6, with an inverted catalytic core sequence), or liposome alone (DOTAP; i.e. N-[l- (2, 3-dioleoyloxy) -N,N,N-trimethylammonium- methylsulfate) .
  • the data are displayed as mean ⁇ SD.
  • FIG 8 shows dose-response experiments for Rs-6 DNAzyme in SMC's. The experimental details are as per Figure 7. The data are expressed as a percentage of the control.
  • Figure 9 shows c-myc expression in DNAzyme-treated SMC's.
  • Cells were labeled with 35 S-methionine as described in Example 7, and immunoprecipitation was performed to determine the expression level of c-myc protein in DNAzyme-treated SMC's.
  • Figure 10 shows the genomic DNA sequence of the human c- myc gene (exons 1 and 2) .
  • This invention is directed to inhibiting the onset of restenosis using DNAzyme technology.
  • the disorder's onset triggered by physical trauma to arterial smooth muscle during angioplasty, is characterized by a several-hour period of c-myc over-expression following shortly thereafter.
  • This c-myc over-expression leads" to excess SMC proliferation, and inhibition of this overexpression in turn inhibits the onset of restenosis.
  • This invention exploits this "window of opportunity" of c-myc over-expression by applying a c- myc mRNA-specific DNAzyme to the area of trauma around the time of angioplasty, thereby cleaving the mRNA and inhibiting restenosis onset.
  • this application provides a DNAzyme which specifically cleaves c-myc mRNA, comprising (a) a catalytic domain that has the nucleotide sequence GGCTAGCTACAACGA and cleaves mRNA at any purine :pyrimidine cleavage site at which it is directed, (b) a binding domain contiguous with the 5' end of the catalytic domain, and (c) another binding domain contiguous with the 3' end of the catalytic domain, wherein the binding domains are complementary to, and therefore hybridize with, the two regions immediately flanking the purine residue of the cleavage site within the c-myc mRNA, respectively, at which DNAzyme-catalyzed cleavage is desired, and wherein each binding domain is at least six nucleotides in length, and both binding domains have a combined total length of at least 14 nucleotides.
  • DNAzyme means a DNA molecule that specifically recognizes and cleaves a distinct target nucleic acid sequence, which can be either DNA or RNA.
  • the instant DNAzyme cleaves RNA molecules, and is of the "10-23" model, as shown in Figure 1, named so for historical reasons. This type of DNAzyme is described in Santoro.
  • the RNA target sequence requirement for the 10-23 DNAzyme is any RNA sequence consisting of
  • the binding domain lengths can be of any permutation, and can be the same or different. Various permutations such as 7+7, 8+8 and 9+9 are envisioned, and are exemplified more fully in the Examples that follow. It is well established that the greater the binding domain length, the more tightly it will bind to its complementary mRNA sequence. According, in the preferred embodiment, each binding domain is nine nucleotides in length. In one embodiment, the instant DNAzyme has the sequence
  • TGAGGGGCAGGCTAGCTACAACGACGTCGTGAC also referred to herein as "Rs-6" .
  • a 3' -3' inversion means the covalent phosphate bonding between the 3' carbons of the terminal nucleotide and its adjacent nucleotide.
  • the instant DNAzymes can contain modified nucleotides.
  • Modified nucleotides include, for example, N3'-P5' phosphoramidate linkages, and peptide-nucleic acid linkages. These are well known in the art (Wagner) .
  • any contiguous purine: pyrimidine nucleotide pair within the c-myc mRNA can serve as a cleavage site.
  • purine ruracil is the desired purine :pyrimidine cleavage site.
  • the c-myc mRNA region containing the cleavage site can be any region.
  • the location within the c-myc mRNA at which DNAzyme-catalyzed cleavage is desired can be the translation initiation site, a splice recognition site, the 5' untranslated region, and the 3' untranslated region.
  • the cleavage site is located at the translation initiation site.
  • n c-myc mRNA means any mRNA sequence encoded by the human c-myc DNA sequence shown in Figure 10 or by any naturally occurring polymorphism thereof.
  • C-myc mRNA includes both mature and immature mRNA.
  • This invention also provides a pharmaceutical composition for inhibiting the onset of restenosis, which comprises the instant DNAzyme and a pharmaceutically acceptable carrier suitable for topical administration.
  • topically administering the instant pharmaceutical composition can be effected or performed using any of the various methods and delivery systems known to those skilled in the art.
  • the topical-- administration can be performed, for example, via catheter and topical injection, and via coated stent as discussed below.
  • compositions for topical administration are well known in the art, as are methods for combining same with active agents to be delivered.
  • the following delivery systems, which employ a number of routinely used carriers, are only representative of the many embodiments envisioned for administering the instant composition.
  • Topical delivery systems include, for example, gels and solutions, and can contain excipients such as solubilizers, permeation enhancers (e.g., fatty acids, fatty acid esters, fatty alcohols and amino acids) , and hydrophilic polymers (e.g., polycarbophil and polyvinylpyrolidone) .
  • the pharmaceutically acceptable carrier is a liposome or a biodegradable polymer.
  • liposomes which can be used in this invention include the following: (1) CellFectin, 1:1.5 (M/M) liposome formulation of the cationic lipid N,N I ,N II ,N III -tetramethyl-N,N I ,N II ,N 111 - tetrapalmitylspermine and dioleoyl phosphatidyl- ethanolamine (DOPE) (GIBCO BRL) ; (2) Cytofectin GSV, 2:1 (M/M) liposome formulation of a cationic lipid and DOPE (Glen Research) ; (3) DOTAP (N- [1- (2, 3-dioleoyloxy) - N,N,N-trimethyl-ammoniummethylsulfate) (Boehringer Manheim) ; and (4) Lipofectamine, 3:1 (M/M) liposome formulation of the polycationic lipid DOSPA and the neutral lipid DOPE (GIBCO BRL) .
  • DOPE dio
  • This invention further provides an angioplastic stent for inhibiting the onset of restenosis, which comprises an agioplastic stent operably coated with a prophylactically effective dose of the instant pharmaceutical composition.
  • Angioplastic stents also known by other terms such as “intravascular stents” or simply “stents”, are well known in the art. They are routinely used to prevent vascular closure due to physical anomalies such as unwanted inward growth of vascular tissue due to surgical trauma. They often have a tubular, expanding lattice-type structure appropriate for their function, and can optionally be biodegradable.
  • the stent can be operably coated with the instant pharmaceutical composition using any suitable means known in the art.
  • "operably coating" a stent means coating it in a way that permits the timely release of the pharmaceutical composition into the surrounding tissue to be treated once the coated stent is administered.
  • Such coating methods for example, can use the polymer polypyrrole. Stents, and methods and compositions for coating same, are discussed in detail in U.S. Serial No. 60/091,217.
  • the prophylactically effective dose contains between about 0.1 mg and about 1 g of the instant DNAzyme. In another embodiment, the prophylactically effective dose contains between about 1 mg and about 100 mg of the instant DNAzyme. In a further embodiment, the prophylactically effective dose contains between about 10 mg and about 50 mg of the instant DNAzyme. In yet a further embodiment, the prophylactically effective dose contains about 25 mg of the instant DNAzyme.
  • This invention further provides a method for inhibiting the onset of restenosis in a subject undergoing angioplasty, which comprises topically administering a prophylactically effective dose of the instant pharmaceutical composition to the subject at around the time of the angioplasty.
  • administering the instant pharmaceutical composition "at around" the time of angioplasy can be performed during the procedure, or immediately before or after the procedure. The administering can be performed according to known methods such as catheter delivery.
  • “Inhibiting" the onset of restenosis means either lessening the severity of restenosis which occurs after angioplasty, or preventing the onset of restenosis entirely. In the preferred embodiment, inhibiting the onset of restenosis means preventing the onset of restenosis entirely.
  • this invention provides a method for inhibiting the onset of restenosis in a subject undergoing angioplasty, which comprises topically administering the instant angioplastic stent to the subject at the time of the angioplasty.
  • the efficacy of DNAzymes in vi tro was determined by measuring the rate of RNA cleavage under multiple turnover conditions. For these experiments, a range of substrate concentrations was used such that [S] ⁇ 10- fold excess over [E] which was fixed at 200 pM.
  • the DNAzyme and a 32p_ ⁇ _ a ]-, e ⁇ e ci synthetic RNA substrate were pre-equilibrated separately for 10 minutes at 37°C in 50 mM Tris.HCl, pH 7.5, 10 mM MgCl2 and 0.01% SDS . At time zero, the reaction was initiated by mixing the
  • the overall catalytic efficiency of each DNAzyme varies significantly between the modified and unmodified species.
  • the inclusion of an inverted base modification produced a 3-fold decrease in the k ca t/K m .
  • the relative efficiency of the long (9+9 bp) arm version was enhanced 10-fold by the presence of an inverted base modification.
  • the intermediate length (8+8 bp) binding arm DNAzyme was the least effected by modification, showing a 2-fold increase in the value of k ca t/K m .
  • the effect of the 3' inverted terminal base was therefore different depending on the length of the substrate-binding arms.
  • the modification was found to be detrimental to the catalytic efficiency.
  • the long (9+9 bp) arm molecule it actually improved catalytic activity.
  • the unmodified DNAzyme activity was optimal with 8 bp substrate binding arms.
  • the overall efficiency was lower due mainly to a higher K m (3.4-23 nM) .
  • a full-length c-myc mRNA was used to further test DNAzymes' ability to cleave various forms of c-myc mRNA under simulated physiological conditions (10 mM MgCl2, pH7.5, 37°C) . Cleavage reactions were performed under 5 single turnover conditions by using 10 nM of long substrate ⁇ c-myc mRNA) and 50 nM of DNAzymes.
  • Figure 5 shows that all the DNAzymes effectively cleave c-myc mRNA with a cleavage rate of 20 to 50%. As expected, the DNAzymes with longer arms cleave substrates more efficiently.
  • a 3 '-inverted base modification decreases the cleavage efficiency of the 7+7 arm DNAzyme, but increases the cleavage efficiency of the 9+9 arm DNAzyme.
  • Anti-c-myc DNAzyme activity was tested in vascular SV40LT (Simian Virus 40 large T antigen) smooth muscle cells (Simons). After growth arrest in 0.5% FBS-DMEM, SMC's were released from Go by addition of 10% FBS- DMEM. Cells were simultaneously exposed to DNAzyme or control oligonuceotide (i.e., the 9/9 arm DNAzyme with an inverted catalytic core sequence) delivered by
  • DNAzymes The impact of DNAzymes on SMC proliferation was also assessed using two independent techniques, i.e., DNA cell-cycle analysis and the determination of mitotic index.
  • DNA histograms were generated at 72 hours after serum stimulation. After this 72-hour interval, 74% of unstimulated cells remained in G 0 /G ⁇ , as compared with only 65% of stimulated cells.
  • Rs-6 the proportion of stimulated cells remaining in G ⁇ /G ⁇ phase increased to 71%.
  • the inactivated DNAzyme control Rs-8 had no effect on the SMC cycle.

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Abstract

La présente invention concerne Un DNAzyme qui clive spécifiquement l'ARNm c-myc, comprenant un domaine catalytique à 15 nucléotides et deux domaines de liaison, ces deux domaines de liaison étant contigus l'un avec l'extrémité 5' du domaine catalytique, l'autre avec l'extrémité 3' du domaine catalytique. Cette invention concerne également une composition pharmaceutique qui est conçue pour empêcher l'apparition de la resténose et qui comprend le DNAzyme susmentionné ainsi qu'un vecteur pharmaceutiquement acceptable convenant pour une administration locale. L'invention porte également sur un stent pour angioplastie destiné à empêcher l'apparition de la resténose, stent qui est enduit d'une dose thérapeutiquement efficace de ladite composition pharmaceutique. Enfin, cette invention concerne des méthodes permettant d'enrayer l'apparition de la resténose chez un patient soumis à une angioplastie et qui consistent soit dans l'administration de la composition pharmaceutique selon l'invention, soit dans la mise en place du stent pour angioplastie chez ce patient.
PCT/IB1999/001484 1998-08-13 1999-08-12 Dnazymes et methodes de traitement de la restenose Ceased WO2000009672A1 (fr)

Priority Applications (5)

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KR1020017001893A KR20010072475A (ko) 1998-08-13 1999-08-12 재발협착증을 치료하기 위한 ｄｎａ자임 및 치료 방법
JP2000565109A JP2002525037A (ja) 1998-08-13 1999-08-12 再狭窄を治療するためのｄｎａザイムおよび方法
AU52984/99A AU5298499A (en) 1998-08-13 1999-08-12 Dnazymes and methods for treating restenosis
CA002340322A CA2340322A1 (fr) 1998-08-13 1999-08-12 Dnazymes et methodes de traitement de la restenose
EP99938479A EP1117768A4 (fr) 1998-08-13 1999-08-12 Dnazymes et methodes de traitement de la restenose

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US9637498P 1998-08-13 1998-08-13
US60/096,374 1998-08-13

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US5807718A (en) * 1994-12-02 1998-09-15 The Scripps Research Institute Enzymatic DNA molecules
WO1998049346A1 (fr) * 1997-04-29 1998-11-05 The Scripps Research Institute Molecules d'adn enzymatiques
DE69940903D1 (de) * 1998-03-27 2009-07-02 Johnson & Johnson Res Pty Ltd Diagnostische methoden basierend auf katalytischen nukleinsäuren

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CN1323344A (zh) 2001-11-21
EP1117768A4 (fr) 2003-09-03
JP2002525037A (ja) 2002-08-13
AU5298499A (en) 2000-03-06
KR20010072475A (ko) 2001-07-31
ZA200101188B (en) 2002-05-13
CA2340322A1 (fr) 2000-02-24
EP1117768A1 (fr) 2001-07-25

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