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WO1997037013A1 - Ribozymes asymetriques en forme de tete de marteau - Google Patents

Ribozymes asymetriques en forme de tete de marteau Download PDF

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Publication number
WO1997037013A1
WO1997037013A1 PCT/AU1997/000210 AU9700210W WO9737013A1 WO 1997037013 A1 WO1997037013 A1 WO 1997037013A1 AU 9700210 W AU9700210 W AU 9700210W WO 9737013 A1 WO9737013 A1 WO 9737013A1
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Prior art keywords
compound
ribozyme
host cell
nucleotides
cleavage
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PCT/AU1997/000210
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English (en)
Inventor
Philip Hendry
Maxine J. Mccall
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Commonwealth Scientific And Industrial Research Organisation
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Priority to JP09534751A priority Critical patent/JP2000509969A/ja
Priority to AU21450/97A priority patent/AU721758B2/en
Priority to EP97913997A priority patent/EP0902836A4/fr
Publication of WO1997037013A1 publication Critical patent/WO1997037013A1/fr
Priority to US09/156,828 priority patent/US6238917B1/en

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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
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/12—Type of nucleic acid catalytic nucleic acids, e.g. ribozymes
    • C12N2310/121—Hammerhead
    • 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
    • C12N2310/32—Chemical structure of the sugar
    • C12N2310/321—2'-O-R Modification
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/35—Nature of the modification
    • C12N2310/351—Conjugate

Definitions

  • cleavage step has been assumed to be essentially independent of the length and sequence of the helices I and III (Fedor, et al . 1992) .
  • the inventors have made a number of observations which suggest that the cleavage rate constant may be dependent of the length and/or sequence of the helices I and III.
  • This invention is directed to a compound having the formula:
  • the compound may be covalently linked to a delivery agent.
  • the invention also includes a composition which comprises the compound in association with an acceptable carrier.
  • the invention also includes a method of cleaving an RNA target sequence which comprises contacting a target sequence with the compound as described above. Further, a method of treating a disease in man or animals associated with a particular RNA which comprises administrating to the man or animal the compound. Further, the invention also includes a diagnostic reagent which comprises the compound.
  • This invention is directed to a compound having the formula: 5'
  • each N represents a nucleotide which may be the same or different and may be substituted or modified in its sugar, base or phosphate provided not every N is a ribonucleotide.
  • the hybridizing arms 3'-(N) n NNNNNNA and NNN(N) n , 5' each represent an oligonucleotide having a predetermined sequence which is complementary to an RNA target sequence to be cleaved.
  • n and n' represents an integer which defines the number of nucleotides in the oligonucleotide with the proviso that n is from 1 to 5 and n' is from 1 to 3 and each * represents base pairing between the nucleotides located on either side thereof.
  • each solid line represents a chemical linkage providing covalent bonds between the nucleotides located on either side thereof.
  • the symbol "a" represents an integer which defines a number of nucleotides with the proviso that a may be 0 or 1 and if 0, the A located 5' of (N) a is bonded to the N located 3' of (N) a .
  • each m and rrt' represents an integer which is greater than 2; wherein P represents a non-nucleotide linker or a nucleotide linker (N) b and wherein (N ⁇ represents an oligonucleotide which may be present with the proviso that b represents an integer which is greater than or equal to 3.
  • the oligonucleotide3'-(N) n NNNNNNA is 3 '-(N,) NNNNNCA.
  • (N) a is absent.
  • In the compound of the integer b of (N) b may be equal to 4 and each of m and m' may be 4 .
  • each N in the compound may be a deoxyribonucleotide.
  • each N of 3' (N) n NNNNNNA and N TN(N) ' 5' may be a deoxyribonucleotide.
  • several of the nucleotides N, A, C, G or U are O methyl or 0-alkyl ribonucleotides.
  • the RNA target sequence for the compound may be a viral RNA target sequence.
  • the compound described above may be covalently linked to a delivery agent.
  • the delivery agent is a peptide, a peptide mimic, a cholesterol, a steroid, a cholesterol derivative, a fat, a vitamin, biotin, folic acid, retinoic acid, a protein, ferritin, LDL, insulin, an antibody, a sugar or an oligosaccharide, polyethylene glycol or a homopolymer or co-polymer of aminoacids.
  • the invention also includes a composition which comprises a compound described above in association with an acceptable carrier.
  • the acceptable carrier may be a cationic lipid, a cholesterol, a cholesterol derivative, a liposome, or a homopolymer or co-polymer of aminoacids.
  • the invention also included a host cell comprising the compound above which may be a prokaryotic host cell or an eukaryotic host cell.
  • the prokaryotic host may be an E. coli host cell.
  • the eukaryotic host cell may be a monkey COS host cell, a Chinese hamster ovary host cell, a mammalian host cell, a plant host cell or yeast cell .
  • the invention also includes a method of cleaving an RNA target sequence which comprises contacting the target sequence with the compound as described above. Further a method of treating a disease in man or animals associated with a particular RNA which comprise ⁇ administrating to the man or animal the compound. Further, the invention also includes a diagnostic reagent which comprises the compound.
  • RNA RNA-armed ribozymes. Similar results are obtained when the length of the helices I and III are limited by the length of the hybridizing arms of the ribozymes; a symmetric 21-mer substrate is cleaved more than 100 fold faster by an asymmetric ribozyme with 5 hybridizing nucleotides in its 5' arm and 10 hybridizing bases in its 3' arm than the converse (10/5) asymmetric ribozyme.
  • Preferred cleavage sites in the target RNA have the sequence "UH,” preferably GUC, GUU, GUA, UUA and UUC.
  • suitable reaction conditions may comprise a temperature from about 4 degree (s) C.
  • nucleotides of the sequences 3 '-(N) n NNNNNNA and NNN NI) 5' of the compounds above may be of any number and sequence sufficient to enable hybridization with the nucleotides in the target RNA, as described herein.
  • these compounds may be covalently attached to an antisense molecule which may be 10 to 100 bases in length.
  • Antisense sequences capable of hybridizing to an RNA in a mammal or plant are well known see (Shewmaker et al . U.S. Patent No. 5,107,065, issued April 21, 1992) .
  • the ribozyme acts as an enzyme, showing turnover, the ratio of ribozyme to substrate may vary widely.
  • a target RNA containing a suitable cleavage site such as UH site may be incubated with the compound described above.
  • the nucleotide sequences 3'-(N) n NNNNNNA and NNN(N) n , 5' of the compounds above are selected to hybridize with their substrate. They may be selected so as to be complementary to nucleotide sequences flanking the cleavage site in the target RNA.
  • an enzyme/substrate complex is formed as a result of base pairing between corresponding nucleotides in the ribozyme and the substrate.
  • Nucleotide sequences complementary to 3'-(N) n NNNNNNA and NNN(N) n , 5' of the compounds above flanking the cleavage site in the substrate may form a double stranded duplex through base pairing.
  • This base pairing is well known in the art [See for example: Sambrook, 1989] .
  • the formation of a double stranded duplex between the nucleotides may be referred to as hybridization [Sambrook, 1989] .
  • the extent of hybridization or duplex formation between the ribozyme and its substrate can be readily assessed, for example, by labeling one or both components, such as with a radiolabel, and then subjecting the reaction mixture to polyacrylamide gel electrophoresis under non-denaturing conditions
  • the compounds are complementary with their target. If the target is cleaved specifically on incubation with the compound, the compound is active and falls within the scope of this invention. Accordingly, a ribozyme containing substituted or modified nucleotides in the conserved region may be simply tested for endonuclease activity in a routine manner.
  • cleavage of a target RNA may be readily assessed by various methods well known in the art [See for example: Sambrook, 1989] .
  • Cleavage may, for example, be assessed by running the reaction products (where the substrate is radioactively labeled) on acrylamide, agarose, or other gel systems under denaturing conditions, and then subjecting the gel to autoradiography or other analytical technique to detect cleavage fragments [Sambrook, 1989] .
  • the invention provides a composition which comprises the compounds above in association with an acceptable carrier.
  • the invention also provides a host cell containing the compounds above which may be a prokaryotic host cell or an eukaryotic host cell e.g. yeast cell or yeast protoplast, E. coli host cell, a monkey host cell
  • COS e.g. COS
  • a Chinese hamster ovary host cell e.g. COS
  • a mammalian host cell e.g. COS
  • a plant host cell e.g. COS
  • a plant protoplast host cell e.g. COS
  • the composition in association with an acceptable carrier.
  • This invention also provides a composition as discussed hereinabove wherein the oligonucleotide is an RNA-DNA hybrid molecule comprising nucleotides which may be substituted or modified in their sugar, base or phosphate group. It is preferred that the oligonucleotide be a hybrid RNA-DNA molecule. However, other substitutions or modifications in the nucleotide are possible providing that endonuclease activity i ⁇ not lost. Such derivatives or modifications are described below.
  • the oligonucleotide compound may co pri ⁇ e deoxyribonucleotides, ribonucleotides , deoxyribonucleotide ribonucleotide hybrids, or nucleotides modified in the sugar, phosphate or base, or an oligonucleotide comprising any mixture thereof, derivatives thereof as herein described.
  • the flanking sequences 3'-(N) n NNNNNNA and NNN(N) n , 5' may be chosen to optimize stability of the ribozyme from degradation. For example, deoxyribonucleotides are resistant to the action of ribonucleases.
  • Modified ba ⁇ es, sugars or phosphate linkages of nucleotides may also provide resistance to nuclease attack. Binding affinity may also be optimized in particular circumstances, by providing nucleotides solely in the form of ribonucleotide ⁇ , deoxyribonucleotides, or combinations thereof.
  • RNA cleavage activity of ribozymes having flanking nucleotide sequences which hybridize to target sequences may be comprised wholly of deoxyribonucleotides.
  • optimization may involve providing a mixture of deoxyribonucleotides and ribonucleotides m the nucleotide sequences 3 '-(N) n NNNNNNA and NNN(N ⁇ n , 5' .
  • nucleotides in the ribozyme which are proximal to the cleavage site in a target RNA may be ribonucleotides .
  • the respective 3' and 5' termini of the sequences 3'-(N) n NNNNNNA and NNN(N) n , 5', or alternatively the 3' and 5' end termini of the ribozyme may be modified to stabilize the ribozyme from degradation.
  • blocking groups may be added to prevent exonuclease attack, in particular 3' -5' progressive exonuclease activity.
  • blocking groups may be selected from substituted or unsubstituted alkyl, substituted or unsubstituted phenyl, substituted or unsubstituted alkanoyl .
  • Substituents may be selected from Cj - C 5 alkyl; halogens such as F, Cl or Br; hydroxy; amino; C x - C 5 alkoxy and the like.
  • nucleotide analogues such as phosphorothioates , methylphosphonates or phosphoramidates or nucleoside derivatives (such as alpha - ano er of the ribose moiety) which are resistant to nuclease attack may be employed as terminal blocking groups.
  • the blocking group may be an inverted linkage such as a 3'-3' thymidine linkage, 3-3' abasic ribose or deoxyribose linkage or a 5' -5' triphosphate linkage as in the guanosine cap.
  • groups which alter the susceptibility of the ribozyme molecule to other nucleases may be inserted into the 3' and/or 5' end of the ribozyme.
  • 9-amino-acridine attached to the ribozyme may act a ⁇ a terminal blocking group to generate resistance to nuclease attack on the ribozyme molecules and/or as an intercalating agent to aid endonucleolytic activity of the ribozyme.
  • spermine or spermidine could be used in a related manner.
  • the compounds of this invention may be covalently or non-covalently associated with affinity agents such as proteins, steroids, hormones, lipids, nucleic acid sequences, intercalating molecules ( ⁇ uch a ⁇ acridine derivatives, for example -amino acridine) or the like to modify binding affinity for a substrate nucleotide sequence or increase affinity for target cells, or localization in cellular compartments or the like.
  • affinity agents such as proteins, steroids, hormones, lipids, nucleic acid sequences, intercalating molecules ( ⁇ uch a ⁇ acridine derivatives, for example -amino acridine) or the like to modify binding affinity for a substrate nucleotide sequence or increase affinity for target cells, or localization in cellular compartments or the like.
  • the ribozymes of the present invention may be as ⁇ ociated with RNA binding peptide ⁇ or proteins which may assist in bringing the ribozyme into juxtaposition with a target nucleic acid such that hybridization
  • Another derivative might be an aptamer which is attached to the compound so that upon the binding to a particular aptamer substrate the ribozyme is activated, in a tissue or cell specific manner (Huizenga et al . 1995) .
  • the aptamer may also serve as an affinity agent to localize the ribozyme to particular tissues, cells or cell-compartment e.g. thrombin (Bracht et al . 1995, 1994) .
  • Nucleotide sequences may be added to the respective 3' and 5' termini of the sequences 3 '-(N) n NNNNNNA and NNN(N) n , 5' or alternatively the 3' and 5' end termini of the ribozyme to increase affinity for substrates. Such additional nucleotide sequences may form triple helices with target sequences [Strobel, 1991] which may enable interaction with an intramolecularly folded substrate.
  • modified bases within the additional nucleotide sequences may be used that will associate with either single stranded or duplex DNA generating base pair, triplet, or quadruplet, interactions with nucleotides in the substrate.
  • Suitable bases would include inosine, 5-methylcytosine, 5-bromouracil and other such bases as are well known in the art, as described, for example, in Principles of Nucleic Acid Structure [Saenger, 1984] .
  • the compound ⁇ of this invention may be produced by nucleotide synthetic techniques which are known in the art, and described for example by Carruthers et al . , Foehler et al . and Sproat et al . [Carruthers, 1987; Foehler, 1986; Sproat, 1984] .
  • Such synthetic procedures involve the sequential coupling of activated and protected nucleotide bases to give a protected nucleotide chain, whereafter protecting groups may be removed by suitable treatment .
  • the compounds will be synthesized on an automated synthesizer such as those made by Applied Biosystems (a Division of Perkin Elmer) , Pharmacia or Millipore.
  • the ribozymes in accordance with this invention may be produced by transcription of nucleotide sequences encoding said ribozymes in host-cells or in cell free systems utilizing enzymes such as T3 , SP6 or T7 RNA-polymerase and made with modified nucleoside triphosphates oligonucleotides or modified after transcription.
  • RNA can be replaced by phosphorothioate linkages by in vi tro transcription using nucleoside 5' -O- (1-thiotriphosphates) .
  • T7 RNA polymerase specifically incorporates the Sp isomer of y-phosphorthiotriphosphate with inversion of configuration to produce the Rp isomer of the phosphorothioate linkage.
  • the methods to produce transcripts fully substituted with phosphorothioate linkages adjacent to a given nucleotide, or to produce partially substituted transcripts containing approximately one phosphorothioate linkage.
  • transcripts fully substituted with phosphorothioate linkages adjacent to a given nucleotide or to produce partially substituted transcripts containing approximately one phosphorothioate linkage per molecule, are described by Ruffner and Uhlenbeck (1990) .
  • Conrad et al . (1995) describe methods of using T7 RNA polymerase to produce chimeric transcripts containing ribonucleotides and deoxyribonucleotides (with and without phosphorothioate linkages) , and also ribonucleotides and 2 ' -O-methylnucleotides (with and without phosphorothioate linkages) .
  • transcripts containing up to 50% deoxyribonucleotides, and up to 58% 2'-0- methylnucleotides have been shown to produce transcripts containing up to 50% deoxyribonucleotides, and up to 58% 2'-0- methylnucleotides.
  • Aurup et al (1992) describe methods for using T7 polymerase to produce transcripts containing 2' -fluoro-2 ' -deoxyuridine, 2' -fluoro-2 ' - deoxycytidine, and 2' -amino-2 ' deoxyuridine. (Aurup, 1992; Conrad, 1995; Ruffner, 1990) . Further methods will be discussed below.
  • Nucleotides represented in the compounds above comprise a sugar, ba ⁇ e, and a monophosphate group or a phosphodiester linkage. Accordingly, nucleotide derivatives or modifications may be made at the level of the sugar, base, monophosphate groupings or phosphodiester linkages. It is preferred that the nucleotides in the compounds above be ribonucleotides or RNA/DNA hybrids, however, other substitutions or modifications in the nucleotide are possible providing that endonuclease activity is not lost .
  • the sugar of the nucleotide may be a ribose or a deoxyribose such that the nucleotide is either a ribonucleotide or a deoxyribonucleotide, respectively.
  • the sugar moiety of the nucleotide may be modified according to well known methods in the art [See for example: Saenger, 1984; Sober, 1970] . This invention embraces various modifications to the sugar moiety of nucleotides as long as such modifications do not abolish cleavage activity of the ribozyme.
  • modified sugars include replacement of secondary hydroxyl groups with halogen, amino or azido groups; 2 ' -alkylation; conformational variants such as the 02' -hydroxyl being cis-oriented to the glycosyl C ⁇ ' -N link to provide arabinonucleosides, and conformational isomers at carbon C x ' to give alpha -nucleosides, and the like. Ring nitrogens may be replaced with carbon such as in 7 deazaguanosine, and z deazadenosine .
  • the invention is directed to compounds with a substituted 2' hydroxyl such as 2' O-allyl, or 2' 0- methyl.
  • the carbon backbone of the sugar may be substituted such as in 2' C-allyl.
  • the base of the nucleotide may be adenine, 2-amino adenine, cytosine, guanine, hypoxanthine, inosine, methyl cytosine, thymine, xanthine, uracil, or another modified base (see below) .
  • Nucleotide bases, deoxynucleotide bases, and ribonucleotide bases are well known in the art and are described, for example in Principles of Nucleic Acid Structure [Saenger, 1984] .
  • nucleotide, ribonucleotide, and deoxyribonucleotide derivatives, substitutions and/or modifications are well known in the art [See for example: Saenger, 1984; Sober, 1970] , and these may be incorporated in the ribozyme made with the proviso that endonuclease activity of the ribozyme is not lost.
  • endoribonuclease activity may be readily and routinely assessed.
  • modified bases are found in nature, and a wide range of modified bases have been synthetically produced [See for example: Saenger, 1984; Sober, 1970] .
  • amino groups and ring nitrogens may be alkylated, such as alkylation of ring nitrogen atoms or carbon atoms such as N- L and N of guanine 5 and C of cytosine; substitution of keto by thioketo groups; saturation of carbon-carbon double bonds, and introduction of a C-glycosyl link in pseudouridine.
  • thioketo derivatives are 6 -mercaptopurine and 6-mercaptoguanine .
  • Bases may be substituted with various groups, such as halogen, hydroxy, amine, alkyl, azido, nitro, phenyl and the like.
  • the phosphate moiety of nucleotide or the phosphodiester linkage of oligonucleotide is also subject to derivatization or modifications, which are well known in the art. For example, replacement of oxygen with nitrogen, sulphur or carbon gives phosphoramidates, (phosphorothioates, phosphorodithioates) and phosphonates, respectively. Substitutions of oxygen with nitrogen, sulphur or carbon derivatives may be made in bridging or non-bridging position ⁇ .
  • a further aspect of the invention provides alternative linkages such as an amide, a sulfonamide, a hydroxylamine, a formacetal, a 3 ' -thiofor acetal, a sulfide, or an ethylene glycol function to replace the conventional phosphodiester linkage. These modifications may increase resistance to cellular nucleases and/or improved pharmacokinetics .
  • Sugar Modifications may be 2' fluoro, 2' amino, 2' O-allyl, 2' C-allyl, 2' 0-methyl, 2' 0-alkyl, 4' -thio-ribose, ⁇ -anomer, arabinose, other sugar ⁇ , or non-circular analogues.
  • Phosphate Modifications may be phosphorothioate
  • non-bridging phosphorodithioate (non-bridging) , 3' bridging phosphorothioate, 5' bridging phosphorothioate, phosphoramidates (including substituted phosphoramidates) , 3' bridging phosphoramidate, 5' bridging phosphoramidate, methyl phosphonate, other alkyl phosphonates or phosphate triesters .
  • the phosphodiester linkage may be replaced by an amide, carbamate, thiocarbamate, urea, amine, hydroxylamine, formacetal, thioformacetal, allyl ether, allyl, ether, thioether, or PNA (peptide nucleic acid) linkage.
  • Modifications in base may be purine, 2 , 6-diaminopurine, 2-aminopurine, 0 6 -methylguanosine, 5-alkenylpyrimidines, 5-propyne pyrimidines, inosine, 5-methylcytosine, pseudouridine. Polymers of monophosphate alkanediols, or other alkanediols.
  • nucleotides may be replaced with the following linkers: 1, 3-propanediol, other alkanediols, or various polymers of ethyleneglycol (e.g. tetraethyleneglycol or hexaethyleneglycol) or abasic ribose or deoxyribose .
  • linkers 1, 3-propanediol, other alkanediols, or various polymers of ethyleneglycol (e.g. tetraethyleneglycol or hexaethyleneglycol) or abasic ribose or deoxyribose .
  • Qther Modification ⁇ The 3' or 5' end may be selected from: 3' -3' inverted linkage (inverted diester or inverted phosphoramidate) .
  • Modified sugars may be synthesized as follows: 2' -deoxy-2 ' -fluoro uridine (Sinha, 1984) ; 2'-deoxy-2' fluoro cytidine (Sinha, 1984) ; 2'-deoxy-2' fluoro adenosine; synthesis and incorporation into ribozyme (Olsen, 1991) ; 2' -deoxy-2' -amino uridine and 2' -deoxy-2' -amino cytidine (Heidenreich, 1994) ; 2' -O-allyl- (uridine or cytidine or adenosine or guanosine) (Available from Boehringer Mannheim, M a n n h e i m , G e r m a n y ) 2' -deoxy-2 ' -C-allyl-ribonucleotide ⁇ (Beigelman et al
  • Modified phosphates may be synthesized as follows: Phosphorothioate; synthesized by modification of oxidation procedure during phosphoramidite synthesi ⁇ .
  • Modified bases may be synthesized as follows: purine; synthesis and incorporation into ribozyme (Slim, 1992; Fu,1992; Fu, 1993) ; 7-deazaguanosine, synthesis and incorporation into ribozyme (Fu, 1993) ; inosine, synthe ⁇ i ⁇ and incorporation into ribozyme (Slim, 1992; Fu, 1993) 7-deazaadenosine, synthesis and incorporation into ribozyme (Fu, 1992; Seela, 1993) .
  • Modifications may be made to the 2 'OH group of the sugar at all non-conserved nucleotides; modifications tested have been 2'H (DNA) , 2'F, 2'amino, 2' -O-allyl, 2' -0-methyl, 2'-C-allyl.
  • the phosphate groups of the non-conserved nucleotides may be phosphorothioates (phosphorothioated DNA or RNA) .
  • non-conserved nucleotides are 5 DNA
  • only two or three phosphates at the 3' and 5' ends of the ribozyme are phosphorothioates.
  • the phosphates 5' to the conserved nucleotides C3 , U4 , G5, G8 and G12, and 3' to A9 and N15.2, may be phosphorothioates; but phosphate ⁇ 5' to A9, A13 and 10 A14 may not be phosphorothioates .
  • Phosphate - 5' phosphate can be phosphorothioate
  • Phosphate - 5' phosphate can be phosphorothioate (Shimayama, 93)
  • Base - 2 -amino group on G base is essential ( cannot be 35 mosine ) (Odai , 1990 ; Fu , 1992 )
  • A6 Base - can be purine (i.e. 6-amino group is not essential) (Fu, 92) .
  • N7 cannot be C7 in A base (Fu,
  • Phosphate - 5' phosphate probably can be phosphorothioate (see N7 phosphate) .
  • Phosphate - 5' phosphate cannot be phosphorothioate
  • 3' phosphate can be phosphorothioate (Shimayama, 1993) .
  • Base - 2-amino group is essential (cannot be inosine)
  • Phosphate - 5' phosphate can be phosphorothioate
  • Phosphate - 5' phosphate cannot be phosphorothioate
  • Base - Can change N7 to carbon (Fu, 1992) .
  • Can be purine (Slim, 1992) .
  • A14 also are 2'F) (Pieken, 1991) .
  • the compounds of the present invention may be prepared by methods known per se in the art for the synthesi ⁇ of RNA olecule ⁇ .
  • the ribozymes of the invention may be prepared from a corresponding DNA sequence (DNA which on tran ⁇ cription yields a ribozyme, and which may be synthesized according to methods known per se in the art for the synthesi ⁇ of DNA) operably linked to an RNA polymerase promoter such as a promoter for T3 or T7 polymerase or SP6 RNA polymerase.
  • the RNA may be subsequently modified or modified nucleotides may be directly incorporated.
  • the compounds may be introduced into cells by electroporation, PEG, high velocity particle bombardment or lipofectants, or introduced into cells by way of micromanipulation techniques such as microinjection, such that the compound enters the host cell .
  • the present invention also includes other means of tran ⁇ fer ⁇ uch a ⁇ genetic bullets (e.g. DNA-coated tungsten particles, high-velocity micro projectile bombardment) and electroporation amongst others
  • the compounds of the present invention have extensive therapeutic and biological applications.
  • disease causing viruses in man and animals may be inactivated by administering to a subject infected with a virus, a compound in accordance with the present invention adapted to hybridize to and cleave specific RNA transcripts of the virus.
  • Such compounds may be delivered by parenteral or other means of administration.
  • the compounds of the present invention have particular application to viral diseases caused for example, by the herpes ⁇ implex virus (HSV) or the human immunodeficiency virus (HIV-1, HIV-2) .
  • HSV herpes ⁇ implex virus
  • HIV-1 human immunodeficiency virus
  • RNAs such as occurs in chronic myeloid leukemia, malignant melanoma or bladder carcinoma pancreatic cancer, acute lymphoblastic leukemia (ALL) , restenosis and acute promyelocytic leukemia (APML or APL) and any disease involving altered patterns of gene expression or that involves the expression of genes that lead to abnormal or deleterious RNAs and/or proteins.
  • ALL acute lymphoblastic leukemia
  • APML or APL acute promyelocytic leukemia
  • the period of treatment would depend on the particular disea ⁇ e being treated and could be readily determined by a phy ⁇ ician. Generally treatment would continue until the di ⁇ ease being treated was ameliorated.
  • the ribozyme ⁇ of the present invention also have particular application to the inactivation of RNA transcripts in bacteria and other prokaryotic cells, animals and yeast cells.
  • RNA and DNA sequences are well known in the art for example as discussed by Cotten and Friedman [Cotten, 1990; Friedman, 1989] .
  • the same widely known methods may be utilized in the present invention.
  • the compounds of this invention may be incorporated into cells by direct cellular uptake, where the ribozymes of this invention would cro ⁇ s the cell membrane or cell wall from the extracellular environment .
  • Agents may be employed to enhance cellular uptake, such as liposomes or lipophilic vehicles, cell permeability agents, ⁇ uch a ⁇ dimethylsulfoxide, and the like.
  • the delivery/targeting molecule may be for example: RNA; modified RNA; DNA; modified DNA; peptides; peptide mimetics; steroids, cholesterol and derivatives; other steroids; fats (saturated and partially unsaturated) ; vitamins or mimetics e.g. biotin,- folic acid; retinoic acid; proteins e.g. ferritin, LDL, insulin or specific antibodies; sugars and oligosaccharide ⁇ .
  • the invention include ⁇ linkage to other agents such as avidin, biotin (Partridge et al .
  • the compounds of the present invention may be combined with pharmaceutically and veterinarally acceptable carriers and excipients which are well known in the art, and include carriers such as water, saline, dextrose and various sugar solutions, fatty acids, liposomes, oils, skin penetrating agents, gel forming agents and the like, as de ⁇ cribed for example in Remington's Pharmaceutical Science ⁇ , 17th Edition, Mack Publi ⁇ hing Co., Easton, Pa., Edited by Ostol et al . , which is incorporated herein by reference.
  • carriers such as water, saline, dextrose and various sugar solutions, fatty acids, liposomes, oils, skin penetrating agents, gel forming agents and the like, as de ⁇ cribed for example in Remington's Pharmaceutical Science ⁇ , 17th Edition, Mack Publi ⁇ hing Co., Easton, Pa., Edited by Ostol et al . , which is incorporated herein by reference.
  • the compounds of this invention may be provided in a composition with one or more anti-viral, anti-fungal, anti-bacterial, anti-parasitic, anti-protazoan or antihelminthic agents or the like, for example as described in the Merck Index (1989) 11th Edition, Merck & Co. Inc.
  • Therapeutic composition may involve topical application of ribozyme to the site of disease.
  • ribozymes may be formulated into a cream containing a concentration of 0.1 nM to 100 mM ribozyme, preferably 1 nM to 1 mM. The cream may then be applied to the site of infection over a 1 to 14 day period in order to cause amelioration of symptoms of the infection.
  • effectiveness and toxicity of the ribozymes and formulations involving them may, for example, be tested on an animal model, such as scarified mouse ear, to which virus particles, such as 2 X 10 6 plaque forming units are added.
  • a titer of infectious virus particles in the ear after treatment can then be determined to investigate effectiveness of treatment, amount of ribozyme required and like considerations. Similar investigations in animal models prior to human trials may also be conducted, for example, in respect of the treatment of psoriasis, papilloma disease, cervical preneoplasia, and in diseases such as HIV infection, bacterial or prokaryotic infection, viral infection and various neoplastic conditions, which involve a deleterious RNA species.
  • Composition ⁇ for topical application are generally in the form of creams, where the ribozymes of this invention may be mixed with viscous components.
  • the compounds of this invention may be incorporated into liposo es or other barrier type preparations to shield the ribozymes from nuclease attack or other degradative agents ( ⁇ uch a ⁇ adverse environmental condition ⁇ such as UV light) .
  • compositions may be provided as unit dosage ⁇ , such as capsules (for example gelatin capsules) , tablets, suppositories and the like.
  • injectable compositions may be in the form of sterile solutions of ribozyme in saline, dextrose or other media which may be buffers or contain stabilizers, antioxidants or similar agents.
  • Compositions for oral administration may be in the form of suspensions, solutions, syrup ⁇ , capsules, tablets and the like.
  • Ribozymes may also be provided in the form of an article for sustained release, impregnated bandages, patches and the like.
  • the compounds of this invention may be embedded in liposomes or biodegradable polymers such as polylactic acid.
  • the compounds described herein may also be used as diagnostics .
  • the compounds above may be designed to detect a particular genetic defect or as RNA restriction enzymes or for gene mapping. By running two reactions in parallel, one in contact with the catalytically active compound above one may be able to detect a genetic mutation, deletion or translocation.
  • the method may be accompanied by an amplification step either the reaction or after the reaction with the catalytic compound.
  • the amplification may be based upon polymerase chain reaction (PCR) , Q-beta replicase (see Stefano, J.E. U.S. Patent No. 5,472,840) ; ligase chain reaction (Kramer et al .
  • WO 94/16105 reverse transcriptase PCR
  • RT-PCR reverse transcriptase PCR
  • 3SR self sustained sequence replication
  • NASBA nucleic acid sequence based amplification
  • LAT ligation activated transcription
  • LCR liga ⁇ e chain reaction
  • RCR repair chain reaction
  • the cleavage kinetics of a number of substrates by various hammerhead ribozymes were investigated.
  • the sequences of the substrate molecules are taken from naturally occurring mRNAs and are identified by their origin.
  • the GH series are derived from a sequence found in rat growth hormone mRNA
  • the TAT series are from the TAT mRNA of HIV-1
  • the Kr series are derived from the mRNA of the Kruppel gene of Drosophila melanogaster .
  • the ribozymes and sub ⁇ trates with the prefix OU are similar to, and in some cases identical to the well- characterized hammerhead entitled HH16 (Hertel et al . , 1994; Hertel et al. , 1996) .
  • the two series of substrates for the OU ribozymes are prefixed OU S, and OU CS.
  • the OU S serie ⁇ contain a guanine ribonucleotide at the 13th position from the 5' end, complexed to the OU ribozymes this forms a G-U base pair.
  • the OU CS (Complementary Substrate) series contains an adenine ribonucleotide in that position, thus forming a Watson-Crick A-U pair in complex with the OU ribozymes .
  • Ribozymes are denoted by an R following the identifying prefix, and substrates by the letter S which are further identified by a number denoting their length in nucleotides, e.g.. S13.
  • Ribozymes A and B There are two versions of hammerhead ribozyme used in this paper, and they are denoted as ribozymes A and B .
  • Ribozymes A (RA) are composed solely of RNA (with the exception of the 3' nucleotide) whereas ribozymes B (RB) possess DNA in the arms that hybridise to the sub ⁇ trate, with the exception of nucleotide ⁇ 15.1 and 15.2 which remain as RNA ( Figure 1) .
  • Kr S17-10/6 is a 17 mer substrate with 10 nucleotides 5' of the C 17 and 6 nucleotides to the 3' side, and is the substate complementary to the ribozyme Kr RA-6/10 ( Figure 2) .
  • Oligonucleotides were synthesized using an Applied Biosystems (Foster City, CA) model 391 DNA synthe ⁇ izer. Oligonucleotide ⁇ with the prefix OU were prepapred using reagent ⁇ obtained from Perkin Elmer (Applied Bio ⁇ y ⁇ tems Division, Foster City, CA) . Protected DNA phosphoramidite monomers were from Millipore (Bedford, MA) or Auspep (Melbourne, Australia) . RNA monomers, protected at the 2' -hydroxyl with tert-butyldimethylsilyl groups (tBDMS) , were from the same sources.
  • tBDMS tert-butyldimethylsilyl groups
  • oligonucleotides For convenience in the syntheses, all the oligonucleotides have a deoxyribonucleotide at their 3' end. Deprotection and purification of oligonucleotides were as described previously (McCall, et al . , 1992) , with the exception that the removal of the t-BDMS group from the 2' position was achieved with the use of neat triethylamine trihydroflouride for 24 hours at room temperature, followed by precipitation of the oligonucleotide with 10 volumes of 1-butanol prior to gel purification.
  • each oligonucleotide was checked by labeling its 5' -end with "P phosphate using T4 polynucleotide kinase (New England Biolabs, Beverly MA USA) and ⁇ - 32 P ATP (Bresatec, Sydney, S.A., Australia) , electrophoresing the molecules on a 10 or 15 % polyacrylamide gel containing 7M urea, and visualizing the molecules by autoradiography or using a Molecular Dynamics Phosphorlmaging sy ⁇ tem (Sunnyvale, CA) ; all oligonucleotides were at least 98% pure as judged by this as ⁇ ay.
  • the concentrations of the purified oligonucleotides were determined by UV spectroscopy using the following molar extinction coefficients for the various nucleotides at 260 nm: A, 15.4 X 10 3 ; G, 11.7 X IO 3 ; C, 7.3 X 10 3 ; T/U, 8.8 X 10 3 Lmol " ' cm ' l . All oligonucleotides were stored in distilled, deionized and autoclaved water at -20°C.
  • oligonucleotides used in this study are as follows. Capital letters refer to ribonucleotides, lower-case letters refer to deoxyribonucleotides.
  • GH Growth Hormone System
  • Kr RA-10/10 5' CUCCAGUGUG CUGAUGA GUCC UUUU GGAC GAAAC UCGCAAAt 3' ; Kr RB-10/10, 5' ctccagtgtg CUGAUGA GUCC UUUU GGAC GAAAC tcgcaaat 3' ; Kr RA-6/10, 5' AGUGUG CUGAUGA GUCC UUUU GGAC GAAAC UCGCAAAt 3' ; Kr S21-10/10, 5' AUU UGC GAG UCC ACA CUG GAg 3' ; Kr S18-10/7; 5' AUU UGC GAG UCC ACA CUg 3' ; Kr S17-10/6; 5' AUU UGC GAG UCC ACA Ct 3' ; Kr S16-10/5, 5' AUU UGC GAG UCC ACA Ct 3' ; Kr S16-10/5, 5' AUU UGC GAG UCC ACA c 3
  • TAT System TAT RA-lO/10, 5' GUCCUAGGCU CUGAUGA GUCC UUUU GGAC GAAAC UUCCUGGa 3' ; TAT RA-6/6, 5' UAGGCU CUGAUGA GUCC UUUU GGAC GAAAC UUCc 3; TAT RB-10/10, 5' gtcctaggct CUGAUGA GUCC UUUU GGAC GAAAC ttcctgga 3' ; TAT RA-10/5, 5' GUCCUAGGCU CUGAUGA GUCC UUUU GGAC GAAAC UUc 3; TAT RA-5/10, 5' AGGCU CUGAUGA GUCC UUUU GGAC GAAAC UUCCUGGa 3' ; TAT S13-6/6, 5' GGAAGUCAGCCUa 3', TAT S21-10/10, 5' TCC AGG AAG UCA GCC UAG GAc 3'
  • the kinetic experiments were performed at 37°C with ribozyme and substrate (labeled at the 5' end with 3 P-phosphate) in 10 mM MgCl 2 and 50 M buffer (Tris or Mes) , using the following procedure.
  • the substrate concentrations were kept very high to ensure complete complex formation and were in the range 2-4 ⁇ M (typically 2 ⁇ M) and the ribozyme concentration was at least 1.5 times that of the substrate (typically 3 ⁇ M) .
  • the ribozyme and substrate together in buffer were pre-treated by heating to 85°C for 2 minutes, centrifuging briefly, and then placing at the reaction temperature for a few minutes. The reaction was initiated by the addition of MgCl 2 .
  • Samples were removed at various time intervals and quenched by addition to two volumes of gel loading buffer containing 80% formamide and 20 mM EDTA.
  • the fraction of substrate cleaved in each sample was determined by separation of the substrate from the 5' -product in a 15% polyacrylamide gel containing 7 M urea, and quantifying the amounts of each using a Molecular Dynamics Phosphorlmaging system and ImageQuant software (Molecular Dynamics, Sunnyvale CA) .
  • the ribozyme and sub ⁇ trate together in buffer were pre-heated to 85°C for 2 minute ⁇ , centrifuged briefly, and then placed at 25°C for a few minute ⁇ .
  • the reaction was initiated by the addition of MgCl 2 .
  • the separation of sub ⁇ trate from the cleavage product ⁇ and kinetic analysis was performed as described previously.
  • Ribozymes with 10 nucleotides in each hybridizing arm form complexes with symmetric 13-nucleotide substrate ⁇ that contain 6 base pair ⁇ in each of helices I and III, while the same ribozymes form complexes with symmetric 21-nucleotide substrates that have 10 base pairs in each of the helices .
  • TAT RA-10/10 such ribozymes cleave 13-mer substrates significantly faster than the corresponding 21-mer substrates (Table 1) .
  • the reactivity of TAT RA-10/lO appears to be anomalous and i ⁇ apparently modulated by some spurious base pairing (see discussion) .
  • TAT and Kr substrates were prepared with varying numbers of nucleotides to the 3' side of the cleavage site, and a constant 10 nucleotides to the 5' side. Rate constants for cleavage of the substrates by TAT RB-10/10, Kr RA-10/10 and Kr RB-lO/10 were measured at a number of pHs ( Figures 3,4,5) . These data show that, with a helix III of 10 base pairs, the optimum length of helix I is 5 ⁇ 1 base pairs.
  • Table 4 shows the rate constants for cleavage of substrates S17-10/6 and S13-6/6 by three ribozymes, TAT RB-10/lO, Kr RA-10/10 and Kr RB-10/lO, in which the ribozyme-substrate complexes have either 10 or 6 base pairs in helix III, while there is a constant 6 base pairs in helix I.
  • the difference in rate constants is only two-fold at most.
  • the effect of the length of helix III on cleavage rate constants is much less pronounced than the effect of the length of helix I.
  • This model of ribozyme cleavage is also able to account for the observation that sub ⁇ titution of DNA for RNA in the hybridising arms of the ribozyme (Hendry, et al . 1992; Shimayama, et al . 1993) or in portions of the substrate (Shimayama, 1994) apparently increases the rate constant for the cleavage step in some circumstances .
  • helix I needs to be relatively unstable and helix III needs to be more stable.
  • helix I was able to be reduced to as few a ⁇ 3 base pairs without loss of activity (Tabler, et al . 1994) ; however, in that case, despite the fact that the ribozyme and substrate were pre-annealed, the observed rate constants were only about 0.01 min -1 at pH 8, 20 mM MgCl 2 , which is about 1000-fold slower than would be expected for efficient cleavage of ⁇ hort substrates.
  • mismatches in either of helix I or III close to the conserved domain were shown to cause a dramatic decrease in activity, but mismatches more distal in helix I had only marginal effects (Zoumadakis, et al . 1994) .
  • mismatches close to the core in helix I were tolerated much more than in helix III, where mismatches in any of the 4 inner-most base pairs caused a significant decrease in k 2 .
  • the interactions involve the 4th and 5th nucleotides in the 5' arm of the ribozyme (nucleotides 2.4 and 2.5) and two nucleotides in helix II (nucleotides 11.3 and 11.4) ; in the structure by Scott et al . (Scott, et al . 1995) , the interactions are between nucleotide 2.5 and nucleotide 11.4. Since we have observed that optimum cleavage rates are ob ⁇ erved when helix I i ⁇ 5 ⁇ 1 base pairs in length, it may be that these inter-helix interactions might be an important factor in the cleavage mechanism. If this is the case, the optimum cleavage rates should always be observed with helix I of about 5 nucleotides irrespective of the strength of the interaction. Thi ⁇ work is in Progressive ⁇ .
  • the ribozyme TAT RA-10/10 appears to be a special case.
  • This ribozyme displays anomalous activity in that it cleaves it ⁇ 13-mer substrate TAT S13-6/6 more than 40 fold slower than does the shorter analogue TAT RA-6/6 at pH 8.00 (Hendry, et al. 1995) .
  • the source of the anomalous behaviour must lie in the terminal nucleotides of the 5' arm of the ribozyme since TAT RA-10/5 cleaves TAT S13-6/6 with a rate constant of only 0.09 ⁇ 0.01 min "1 at pH 7.13, 50 fold ⁇ lower the cleavage of that substrate by TAT RA-5/10 under the same conditions.
  • terminal nucleotides in the 5' arm of the ribozyme have the base sequence 5' GUCC, which is complementary to 4 bases in helix II, and in addition there is a high degree of complementarity between the sequences
  • HH16 has been extensively studied (Hertel et al. , 1994), including a recent paper on the effect of shortening helix I (Hertel et al . , 1996) .
  • the ribozyme is widely held to be an example of a well understood and well behaved hammerhead in which the cleavage step is rate-determining.
  • HH16 has a G-U mismatch at position 1.4-2.4 in the standard nomenclature. Uhlenbeck' s data did not show the expected (from our point of view) peak in cleavage rate constant when helix 1 was around 5 or 6 nucleotide ⁇ in length.
  • minizymes ribozymes lacking helix II
  • minizymes ribozymes lacking helix II
  • 10 nucleotides in each hybridising arm invariably cleave symmetrical 21-mer sub ⁇ trates more efficiently than symmetrical 13-mer sub ⁇ trate ⁇
  • no improvement in cleavage rate con ⁇ tants is observed for these minizymes cleaving 10/5 substrates compared with 10/10 substrates (where there are 5 and 10 base pairs, respectively, in helices I of the complexes) .
  • conformational flexibility possessed by the minizymes because of their lack of helix II, allows them to attain the active conformation without dissociation of helix I.
  • Denman et al (Denman, et al . 1995) have studied a number of ribozymes targeted to the -amyloid peptide precursor (3-APP) , with the expressed goal of investigating the effect of destabili ⁇ ing helix I. In their study, which was apparently complicated by multiple substrate conformations, the observed cleavage rates were very low. They concluded that the most active ribozyme was one with helices I and III of 7 and 8 base pairs respectively. Amiri and Hagerman (Amiri, et al .
  • the 5' hybridising arm should contain in the order of 5 ⁇ 1 nucleotides.
  • the number of nucleotides in the 3' hybridi ⁇ ing arm is less critical; however there should greater than about 5 nucleotides. If multiple turnovers are required, then the rate of dissociation of the 5' product of cleavage from the 3' arm of the ribozyme (i.e. the rate of dissociation of helix III after cleavage) also must be considered.
  • G5 can't be DNA.
  • A6 and A9 can be DNA.
  • G5 can't be inosine .
  • G8 can be inosine, and A6 and A9 can be purine.
  • U4 or A6, but not both may be 2' -O-allyl, without much loss in activity, but better activity is achieved if U4 and A6 are unmodified RNA.
  • U4, G5, A6, G8 and A15.1 as RNA and other nucleotide ⁇ 2' -O-allyl, G12 can be DNA.
  • G12, A13 and A14 reduces activity, but doesn't eliminate it. Making G5 as DNA results in big reduction in activity. ) Perreault, J.-P., Labuda, D., Usman, N., Yang, J.-H. and Cedergren, R. (1991) , Biochemistry, 30: 020-4025.
  • G5 and A9 cannot be DNA.
  • G12, A13 , A14, G8 can be
  • G5 and G8 cannnot have 2'F, 2'H, or 2 ' amino (reduction in k(cat) i ⁇ about 150-fold for 2'F, 2'H, and about 10-fold for
  • G12 can have 2'H and 2'amino, but cannot have

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Abstract

L'invention concerne un composé ayant la formule (I). Dans cette formule, N peut être un nucléotide, n est égal à 1-5 et n' est égal à 1-3. Ces ribozymes asymétriques présentent des vitesses de scission plus élevées que la normale. Le composé peut être lié par covalence à un agent d'administration. L'invention concerne, en outre, une composition qui comprend le composé en association avec un vecteur acceptable. L'invention concerne également une méthode pour scinder une séquence cible d'ARN avec le composé décrit ci-dessus. L'invention concerne, également, une méthode pour traiter une maladie associée à un ARN particulier, chez un humain ou chez un animal, qui consiste à administrer le composé à cet humain ou à cet animal. En outre, l'invention concerne un réactif de diagnostic qui comprend le composé.
PCT/AU1997/000210 1996-04-02 1997-04-02 Ribozymes asymetriques en forme de tete de marteau WO1997037013A1 (fr)

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EP97913997A EP0902836A4 (fr) 1996-04-02 1997-04-02 Ribozymes asymetriques en forme de tete de marteau
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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001016312A3 (fr) * 1999-08-31 2001-08-09 Ribozyme Pharm Inc Modulateurs d'expression genetique a base d'acide nucleique
WO2001030362A3 (fr) * 1999-10-26 2002-01-17 Immusol Inc Therapie ribozymique destinee au traitement de maladies proliferantes de la peau et des yeux
WO2004038019A3 (fr) * 2002-10-23 2004-08-19 Isis Innovation Dnazyme

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1993023569A1 (fr) * 1992-05-11 1993-11-25 Ribozyme Pharmaceuticals, Inc. Procede et reactif d'inhibition de la replication virale
WO1993023057A1 (fr) * 1992-05-14 1993-11-25 Ribozyme Pharmaceuticals, Inc. Procede et reactif destines a empecher l'evolution du cancer
WO1994002595A1 (fr) * 1992-07-17 1994-02-03 Ribozyme Pharmaceuticals, Inc. Procede et reactif pour le traitement de maladies chez les animaux
AU7937894A (en) * 1993-10-15 1995-05-04 Dkfz Deutsches Krebsforschungszentrum Stiftung Des Offentlichen Rechts Asymmetric hammerhead ribozymes and nucleotide sequences for their construction

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Publication number Priority date Publication date Assignee Title
CA2084790C (fr) * 1990-06-19 2002-04-09 Philip Anthony Jennings Endonucleases

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1993023569A1 (fr) * 1992-05-11 1993-11-25 Ribozyme Pharmaceuticals, Inc. Procede et reactif d'inhibition de la replication virale
WO1993023057A1 (fr) * 1992-05-14 1993-11-25 Ribozyme Pharmaceuticals, Inc. Procede et reactif destines a empecher l'evolution du cancer
WO1994002595A1 (fr) * 1992-07-17 1994-02-03 Ribozyme Pharmaceuticals, Inc. Procede et reactif pour le traitement de maladies chez les animaux
AU7937894A (en) * 1993-10-15 1995-05-04 Dkfz Deutsches Krebsforschungszentrum Stiftung Des Offentlichen Rechts Asymmetric hammerhead ribozymes and nucleotide sequences for their construction

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Title
See also references of EP0902836A4 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001016312A3 (fr) * 1999-08-31 2001-08-09 Ribozyme Pharm Inc Modulateurs d'expression genetique a base d'acide nucleique
WO2001030362A3 (fr) * 1999-10-26 2002-01-17 Immusol Inc Therapie ribozymique destinee au traitement de maladies proliferantes de la peau et des yeux
WO2004038019A3 (fr) * 2002-10-23 2004-08-19 Isis Innovation Dnazyme

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EP0902836A4 (fr) 2004-12-22
AU2145097A (en) 1997-10-22

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