[go: up one dir, main page]

WO1997038097A1 - Oligonucleotides cooperatifs - Google Patents

Oligonucleotides cooperatifs Download PDF

Info

Publication number
WO1997038097A1
WO1997038097A1 PCT/US1997/005683 US9705683W WO9738097A1 WO 1997038097 A1 WO1997038097 A1 WO 1997038097A1 US 9705683 W US9705683 W US 9705683W WO 9738097 A1 WO9738097 A1 WO 9738097A1
Authority
WO
WIPO (PCT)
Prior art keywords
oligonucleotide
oligonucleotides
nucleic acid
cooperative
composition
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/US1997/005683
Other languages
English (en)
Inventor
Ekambar R. Kandimalla
Sudhir Agrawal
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Aceragen Inc
Original Assignee
Hybridon Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from US08/420,672 external-priority patent/US6372427B1/en
Application filed by Hybridon Inc filed Critical Hybridon Inc
Priority to AU27234/97A priority Critical patent/AU2723497A/en
Publication of WO1997038097A1 publication Critical patent/WO1997038097A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • 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
    • C12N15/1131—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 viruses
    • 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
    • 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
    • C12N2310/50—Physical structure
    • C12N2310/53—Physical structure partially self-complementary or closed

Definitions

  • antisense oligonucleotides utilize highly sequence-specific complementary nucleo-base recognition of target nucleic acids through Watson-Crick hydrogen bonding between A and T, and G and C, that leads to the development of less toxic and more site specific chemotherapeutic agents (Stephenson et al. (1978) Proc. Natl. Acad. Sci. (USA) 75:285-288). As per theoretical calculations, an oligonucleotide of 13 or more bases long should bind to a unique sequence that occurs only once in a eucaryotic mRNA pool.
  • the present invention provides cooperative oligonucleotides with improved sequence specificity for a single-stranded target, reduced toxicity, and improved biological activity as antisense molecules.
  • the present invention provides a composition including at least two synthetic cooperative oligonucleotides, each comprising a region complementary to one of tandem, non-overlapping regions of a target single-stranded nucleic acid, and a dimerization domain at a terminus of each of the oligonucleotides.
  • the dimerization domains of the cooperative oligonucleotides are complementary to each other, and the target nucleic acid being an mRNA, single- stranded viral DNA, or single-stranded viral RNA.
  • the oligonucleotides each are complementary to tandem regions of the target nucleic acid that are separated by 0 to 3 bases. In some preferred embodiments, each of the oligonucleotides are about 9 to 25 nucleotides in length. In one embodiment, the composition consists of two cooperative oligonucleotides, the dimerization domain of a first or one of the oligonucleotides being located at its 3' terminal portion, and being complementary to the dimerization domain of a second or the other oligonucleotide which is located at its
  • the dimerization domain of the first cooperative oligonucleotide is located at its 3' terminal portion, and is complementary to the dimerization domain of a second oligonucleotide which is located at its 3' terminal portion.
  • the dimerization domain of the first cooperative oligonucleotide is located at its 5' terminal portion, and is complementary to a dimerization domain of the second oligonucleotide which is located at its 5' terminal portion.
  • the invention provides in another aspect a duplex structure comprising first and second synthetic cooperative oligonucleotides, each oligonucleotide comprising a region complementary to the non-overlapping, tandem regions of the target nucleic acid which is an mRNA, single-stranded viral RNA, or single-stranded viral DNA.
  • the first oligonucleotide in the duplex has a terminal dimerization domain complementary and hybridized to the dimerization domain of the second oligonucleotide.
  • each of the oligonucleotides are about 9 to 25 nucleotides in length, and in others, the dimerization domains of the first and second oligonucleotides each comprise about 3 to 7 nucleotides.
  • the invention provides first and second oligonucleotides which are complementary to tandem regions of the target nucleic acid separated by 0 to 3 bases.
  • the duplex structure is hybridized to the target nucleic acid.
  • the invention also provides pharmaceutical formulations containing the compositions or duplex structures described above, and methods of inhibiting the expression of a nucleic acid in vitro comprising the step of treating the nucleic acid with the pharmaceutical formulations of the invention.
  • the first and second oligonucleotides are complementary to an HIV DNA or an HIV RNA.
  • the invention provides a ternary structure comprising a first synthetic cooperative oligonucleotide, a second synthetic cooperative oligonucleotide, and a third synthetic cooperative oligonucleotide, each oligonucleotide comprising a region complementary to one of tandem, non-overlapping regions of a target nucleic acid, and each comprising a dimerization domain at one or both of their termini.
  • the dimerization domain of the first oligonucleotide is complementary and hybridized to a first dimerization domain at one terminus of the third oligonucleotide and the dimerization domain of the second oligonucleotide is complementary and hybridized to a second dimerization domain at the other terminus of the third oligonucleotide when the first, second, and third oligonucleotides are hybridized to the target nucleic acid.
  • the target nucleic acid is an mRNA, a single-stranded viral RNA, or a single-stranded viral DNA.
  • the ternary structure is hybridized to the target nucleic acid.
  • the invention provides methods of inhibiting the expression of a nucleic acid comprising the step of contacting the nucleic acid with at least one cooperative oligonucleotide of the invention.
  • FIG. 1A is a schematic representation of the cooperative binding of two short oligonucleotides to tandem sites
  • FIG. 1B is a schematic representation of the binding to adjacent sites on a target nucleic acid of cooperative oligonucleotides that have extended antisense dimerization domains and their dimerization;
  • FIG. 1C is a schematic representation of the binding of three cooperative oligonucleotides of the invention to adjacent sites on a target nucleic acid;
  • FIG. 2A is a graphic representation showing the thermal melting profile (dA/dT vs. T) of oligonucleotides 1-7 shown in Table 2 with their DNA target;
  • FIG. 2B is a graphic representation showing the thermal melting profile (dA/dT vs. T) of oligonucleotides 1+2, 1+3, 1+4, and 5 shown in Table
  • FIG. 3 is a graphic representation showing the thermal melting profiles (dA/dT vs. T) of the oligonucleotide combinations with extended antisense dimerization domains (10+14, 11+15, 9+14, 12+16, and 13+17);
  • FIG. 4A is an autoradiogram showing the RNase H hydrolysis pattern of the RNA target sequence in the presence of oligonucleotides 5, 1, 2, 1+2, 14, 10, and 10+14 at different time points;
  • FIG. 4B is an autoradiogram showing the RNase H hydrolysis pattern of the RNA target sequence in the presence of oligonucleotides 5, 13, 17, and 13+17 at different time points;
  • FIG. 5 is an autoradiogram showing the RNase H hydrolysis pattern of RNA target in the presence of the mismatched oligonucleotides 23, 24, 18 and 19 compared to the control matched oligonucleotide 5 and 1 at different time points;
  • FIG. 6 is a graphic representation showing the ability of cooperative oligonucleotide oligonucleotides 1+2 and 13+17 and
  • FIG. 7 is a graphic representation showing the percent inhibition of HIV-1 in cell cultures by cooperative antisense oligonucleotides 1+2, 13+17, 9+14, 10+14, and 12+16 and by control antisense oligonucleotides 5 and 20, present at two different concentrations;
  • FIG. 8 is a graphic representation showing the relationship between meeting temperature (Tm) and percent HIV-1 inhibition for cooperative oligonucleotides 10+14, 12+16, and 13+17.
  • Cooperative interactions between biological macromolecules are important in nature. For example, the cooperative interactions between proteins and nucleic acids are vital for the regulation of gene expression. Cooperative interactions serve to improve sequence specificity, affinity, and biological activity (Ptashne (1986) A Genetic Switch ; Blackwell Scientific Publications and Cell Press: Palo Alto, CA). Cooperative binding of drugs to DNA (Asseline et al. (1984) Proc. Natl. Acad. Sci. (USA) 81:3297-3301; Rao et al. (1991) J. Org. Chem. 56:786-797), of oligonucleotides or their conjugates to single stranded DNA (Tazawa et al. (1972) J. Mol. Biol. 66:115-130; Maher et al. (1988) Nucl. Acids Res.
  • the present invention provides synthetic oligonucleotides which interact with mRNA, single- stranded viral RNA, or single-stranded viral DNA ("target nucleic acids"), and have improved affinity, specificity, and biological activity as antisense molecules. At least two of the oligonucleotides of the invention are used to interact with a target nucleic acid, thereby enabling them to interact cooperatively, synergistically enhancing their ability (singly) to inhibit expression of the target nucleic acid.
  • synthetic oligonucleotide for purposes of this invention includes chemically synthesized polymers of about 7 to about 25, and preferably from about 9 to about 23 nucleotide monomers (nucleotide bases) connected together or linked by at least one 5' to 3' internucleotide linkage.
  • Some cooperative oligonucleotides of the invention are complementary to non-overlapping, tandem regions of the target nucleic acid, as shown in FIG. 1A, while others' are complementary to adjacent sites (FIGS. 1B and 1C). At least two of these oligonucleotides can used to control target nucleic acid expression.
  • oligonucleotide complementary to a target nucleic acid is intended to mean an oligonucleotide sequence that binds to the nucleic acid sequence under physiological conditions, e.g., by Watson- Crick base pairing (interaction between oligonucleotide and single-stranded nucleic acid in antiparallel orientation) or by Hoogsteen base pairing (interaction between oligonucleotide and double-stranded or single-stranded nucleic acid in parallel orientation) or by any other means including in the case of a oligonucleotide binding to RNA, pseudoknot formation.
  • Watson-Crick base pairing interaction between oligonucleotide and single-stranded nucleic acid in antiparallel orientation
  • Hoogsteen base pairing interaction between oligonucleotide and double-stranded or single-stranded nucleic acid in parallel orientation
  • any other means including in the case of a oligonucleotide binding to
  • the inhibitory ability of the cooperative oligonucleotides of the invention is enhanced even further when these oligonucleotides also include a terminal portion (i.e., a "dimerization domain") which is not complementary to the target nucleic acid, but rather which is complementary to each other, thereby enabling the formation of a dimers (FIG. 1B).
  • a dimerization domain i.e., a dimerization domain
  • the regions of the cooperative oligonucleotides which are complementary to the target nucleic acid may be separated by 0 to 3 bases.
  • the cooperative oligonucleotides of the invention may have any nucleotide sequence, as long as a portion of its sequence is complementary to a portion of a target nucleic acid. Any nucleic acid may be targeted by the cooperative oligonucleotides of the invention including viral, bacterial, and cellular genes, mRNAs, or cDNAs.
  • terminal dimerization domains of cooperative oligonucleotides which form duplexes with each other may not be not complementary to the target nucleic acid. These dimerization domains may be at the 3' termini of both cooperative oligonucleotides, at the 5' termini of both cooperative oligonucleotides, or at the 3' terminus of one cooperative oligonucleotide and the 5' terminus of the other cooperative oligonucleotide.
  • the cooperative oligonucleotides of the invention are composed of deoxyribonucleotides, ribonucleotides, or any combination thereof, with the 5' end of one nucleotide and the 3' end of another nucleotide being covalently linked, in some cases, via a phosphodiester internucleotide linkage.
  • the oligonucleotides can be prepared by art recognized methods such as phosphoramidate, H- phosphonate chemistry, or methylphosphoramidate chemistry (see, e.g., Uhlmann et al. (1990) Chem. Rev. 90:543-584; Agrawal et al. (1987) Tetrahedron. Lett.
  • oligonucleotides of the invention may also be modified in a number of ways, for example, to enhance stability, without compromising their ability to hybridize to nucleotide sequences contained within a targeted region of a particular gene.
  • modified oligonucleotide as used herein describes an oligonucleotide in which at least two of its nucleotides are covalently linked via a synthetic linkage, i.e., a linkage other than a phosphodiester linkage between the 5' end of one nucleotide and the 3' end of another nucleotide in which the 5' nucleotide phosphate has been replaced with any number of chemical groups.
  • oligonucleotides with phosphorothioate linkages can be prepared using methods well known in the field such as methoxyphosphoramidite (see , e.g. , Agrawal et al. (1988) Proc. Natl. Acad.
  • any of these chemical groups or linkages may also be substituted with various other chemical groups, e.g., an aminoalkylphosphonx ate.
  • Oligonucleotides with these chemical groups can be prepared according to known methods (see, e.g., Agrawal and Goodchild
  • alkylphosphonate and/or phosphoramidate and/or phosphotriester internucleoside linkage flanked by one or more region of oligonucleotide phosphorothioate.
  • the phosphorothioate linkages may be mixed Rp and Sp enantiomers, or they may be stereoregular or substantially stereoregular in either Rp or Sp form (see Iyer et al. (1995) Tetrahedron Asymmetry 6:1051-1054).
  • Thiono triesters can be prepared according to the methods described in U.S. Ser. No. 08/409,169, filed March 23, 1993.
  • Oligonucleotides with phosphorothioate linkages can be prepared using methods well known in the field such as phosphoramidite (see, e.g., Agrawal et al.
  • Oligonucleotides which are self-stabilized are also considered to be modified oligonucleotides useful in the methods of the invention (Tang et al. (1993) Nucleic Acids Res . 20 :2729-2735). These oligonucleotides comprise two regions: a target hybridizing region; and a self-complementary region having an oligonucleotide sequence complementary to a nucleic acid sequence that is within the self- stabilized oligonucleotide.
  • modifications include those which are internal or are at the end(s) of the oligonucleotide molecule and include additions to the molecule of the internucleoside phosphate linkages, such as cholesteryl or diamine compounds with varying numbers of carbon residues between the amino groups and terminal ribose, deoxyribose and phosphate modifications which cleave, or crosslink to the opposite chains or to associated enzymes or other proteins which bind to the viral genome.
  • cholesteryl or diamine compounds with varying numbers of carbon residues between the amino groups and terminal ribose, deoxyribose and phosphate modifications which cleave, or crosslink to the opposite chains or to associated enzymes or other proteins which bind to the viral genome.
  • modified oligonucleotides include oligonucleotides with a modified base and/or sugar such as arabinose instead of ribose, or a 3', 5'- substituted oligonucleotide having a sugar which, at both its 3' and 5' positions is attached to a chemical group other than a hydroxyl group (at its
  • modifications to sugars include modifications to the 2' position of the ribose moiety which include but are not limited to
  • WO 94/02498 discloses traditional hybrid oligonucleotides having regions of 2'-O- substituted ribonucleotides flanking a DNA core region.
  • U.S. Patent Application Serial No. 08/516,454, filed August 17, 1995 discloses an "inverted" hybrid oligonucleotide which includes an oligonucleotide comprising a 2'-O-substituted (or 2' OH, unsubstituted) RNA region which is in between two oligodeoxyribonucleotide regions, a structure that "inverted relative to the "traditional" hybrid oligonucleotides.
  • modified oligonucleotides are capped with a nuclease resistance-conferring bulky substituent at their 3' and/or 5' end(s) , or have a substitution in one nonbridging oxygen per nucleotide.
  • Such modifications can be at some or all of the internucleoside linkages, as well as at either or both ends of the oligonucleotide and/or in the interior of the molecule (reviewed in Agrawal et al. (1992) Trends Biotechnol. 10:152-158).
  • oligonucleotides of the invention were prepared as described above and tested for their ability to inhibit the expression of a target gene.
  • the target chosen was a sequence in the initiation codon region of gag mRNA of HIV-1 (SEQ ID NOS:21 and 22) (Agrawal and Tang (1992) Antisense Res. Dev. 2:261). A nonlimiting list of some representative gag oligonucleotides are shown in TABLE 1.
  • Oligonucleotides 1 and 2 are designed to bind to 21 bases of the target nucleic acid at adjacent sites without any base gap between them (see FIG. 1A and TABLE 1). Thus, contact is expected to be maintained through the 3'- end of the oligonucleotide 1 and the 5'-end of the oligonucleotide 2 when these oligonucleotides bind to the target sequence at the adjacent sites. This results in cooperativity in the interactions of these two oligonucleotides.
  • Oligonucleotides 3 (SEQ ID NO: 3) and 4 (SEQ ID NO: 4) bind to the same site as oligonucleotide 2 but are separated by 1 and 2 bases on the target sequence, gaps, respectively, from the binding site of oligonucleotide 1. Because of this gap these oligonucleotides are expected not to show any cooperativity in the binding of these oligonucleotide pairs to the target.
  • Oligonucleotide 5 (SEQ ID NO: 5) binds to the same 21 base target sequence in the target oligonucleotide that oligonucleotides 1 and 2 together bind.
  • Oligonucleotide 6, a 22mer (SEQ ID NO : 6 ) and oligonucleotide 7, a 23mer (SEQ ID NO : 7 ) have 1 and 2 mismatches, respectively, in position that correspond to 1 and 2 base separation when oligonucleotides 1+3 and 1+4 bind to the target sequence together.
  • Oligonucleotide 8 (SEQ ID NO: 8) is a 13mer control oligonucleotide that binds to the same sequence as oligonucleotides 2 and 3 adjacent to oligonucleotide 1 without a base separation between them.
  • oligonucleotides 1 and 2 were both extended at the site of junction with complementary sequences so that they form a duplex stem upon interaction with the target, as shown in FIG. 1B.
  • This extended antisense dimerization domain is designed not to have any complementarity with the adjacent bases of the antisense oligonucleotide binding site on the target.
  • Oligonucleotides 9-17 (SEQ ID NOS: 9-17) have an extended sequence on either the 5'- or 3'-end of the binding sequence, which forms a duplex stem between the two oligonucleotides when they bind to adjacent sites on the target (FIG. 1B).
  • Oligonucleotides 9 and 14 form a 3 base pair stem. Oligonucleotides 10 and 14 have the same length of extended antisense dimerization domain but with one base separating the two target sites of the binding oligonucleotide pair. Oligonucleotide pairs 11+15, 12+16, and 13+17 bind to the same length of the sequence on the target as oligonucleotide pair 10+14 but with 4, 5, and 7 base pair extended antisense dimerization domains, respectively.
  • the initial evidence for cooperative binding of oligonucleotides 1 and 2 to their target sequence comes from thermal melting studies.
  • TABLE 2 shows thermal melting data of the duplexes of these oligonucleotides individually and together with other corresponding oligonucleotides (FIG. 2).
  • the resulting duplex has a Tm of 47.8°C.
  • Duplexes of oligonucleotides 1+3 and 1+4 with the target sequence have Tms of 44.4°C and 46°C, respectively.
  • the oligonucleotides 1 and 3 bind to the target with a 1 base gap between them, and the oligonucleotides 1 and 4 bind to the target with a 2 base gap between them.
  • the Tm of the duplex formed by oligonucleotides 1 and 2 together with the target is more than the average of the duplexes formed by 1 and 2 individually with the target sequence
  • the duplex of oligonucleotide 5 which binds to the entire 21 base length has a Tm of 67.7°C.
  • the duplex of oligonucleotide 6 (SEQ ID NO: 6), a 22-mer with a mismatch in place that corresponds to one base gap between oligonucleotides 1 and 3, has a Tm of 64.2°C.
  • the duplex of oligonucleotide 7 (SEQ ID NO: 7), a 23mer with two mismatches in a position that corresponds to the two base gap between oligonucleotides 1 and 4, has a Tm of 59.9°C.
  • oligonucleotides 6 and 7 which bind to the target with one or two base mismatches indicate that these oligonucleotides can bind to a number of sites other than the perfectly matched target site at physiological temperatures. Thus, sequence specificity is decreasing.
  • duplex stem length results in the formation of a stable complex between the two tandem oligonucleotides in the absence of the target sequence, an occurrence which is not desirable. In all the cases, a sharp cooperative single melting transition was observed
  • Modified cooperative oligonucleotides were studied for their antisense abilities. For example, phosphorothioate internucleotide-linked forms of cooperative oligonucleotides were studied for their ability to activate RNase H.
  • RNase H is an enzyme that recognizes RNA-DNA heteroduplexes and hydrolyses the RNA component of the heteroduplex (Cedergren et al. (1987) Biochem. Cell Biol. 65:677).
  • Some studies have shown that antisense oligonucleotides have less transition inhibition activity in RNase H-free systems than in systems where RNase H is present (Haeuptle et al. (1986) Nucleic Acids Res. 14:1427-14448; Minshull et al.
  • RNA target sequence (SEQ ID NO: 22) which encodes a portion of the HIV-1 gag gene (TABLE 1) was synthesized to study the RNase H activation property of modified cooperative oligonucleotides of the invention.
  • modified oligonucleotides 1, 10, and 17 bind to a 9 base site on the 3 '-side of the binding site of the target, and modified oligonucleotides 2, 13, and 14 bind on the 5 -side of the target adjacent to the binding site of the former oligonucleotide.
  • Oligonucleotide 5 binds to the entire length of the 21 bases on the target.
  • Oligonucleotides 6, 7, 18 and 19 contained mismatches.
  • FIGS. 4A and 4B An autoradiogram showing the RNase H hydrolysis pattern of the RNA target in the absence and presence of oligonucleotides of the invention is shown in FIGS. 4A and 4B.
  • hydrolytic activity is observed towards the 3'-end of the target RNA (lower half of the autoradiogram) in which oligonucleotides 1, 14, and 17, respectively, are present.
  • RNA degradation bands are present only in the upper half of the autoradiogram, indicating the binding of oligonucleotides 2, 10, and 13, respectively, on the 5'-side of the target.
  • oligonucleotide 23 Two short oligonucleotides analogous to oligonucleotide 1 having one and two mismatches, oligonucleotides 18 (SEQ ID NO: 18) and 19 (SEQ ID NO: 19), were synthesized and studied for RNase H activation in comparison to oligonucleotides 23 and 24.
  • FIG. 5 shows the RNase H hydrolytic pattern of target RNA in the presence of the mismatched oligonucleotides.
  • Oligonucleotide 23 Two short oligonucleotides analogous to oligonucleotide 1 having one and two mismatches, oligonucleotides 18 (SEQ ID NO: 18) and 19 (SEQ ID NO: 19), were synthesized and studied for RNase H activation in comparison to oligonucleotides 23 and 24.
  • FIG. 5 shows the RNase H hydrolytic pattern of target RNA in the presence of the mismatched oligonucleotides
  • Oligonucleotide 18 with one mismatch shows little or no RNA degradation compared to oligonucleotide 1 (experiment 4). However, it appears that oligonucleotide 18 has a strong binding site on the 5'-end of the RNA target as indicated by the RNA degradation bands towards the 5'-end of the RNA. No digestion of the 3'-end of the RNA target and little digestion of the 5'-end was observed with oligonucleotide 19, which has two mismatches (experiment 6). This clearly demonstrates that the new cooperative oligonucleotides bind with sequence specifically.
  • Representative modified cooperative oligonucleotides of the invention were also studied for their HIV-1 virus inhibition properties in cell cultures.
  • the results using phosphorothioate cooperative oligonucleotides are shown in FIG. 6 as a graph of percent virus inhibition versus concentration of the oligonucleotide (s) and FIG. 7.
  • the combination of oligonucleotides 1+2, which bind to the same sequence on the target as oligonucleotide 5 also failed to show much activity.
  • IC 50 for oligonucleotide 20 in the same assay system was about 0.55 ⁇ M .
  • oligonucleotide combination 13+17 which forms a 7 base pair dimerization duplex stem.
  • This oligonucleotide combination showed activity close to oligonucleotide 20, with an IC 50 value of about 4.0 ⁇ M.
  • the combination 10+4 which forms a three base pair extended dimerization stem, showed about 15% virus inhibition at 4 ⁇ M concentration
  • oligonucleotide 17 when it was tested alone, was 4 ⁇ M .
  • the other oligonucleotides studied individually or in combinations did not show significant activity even up to 10 ⁇ M concentration (FIG. 7).
  • the oligonucleotides 9+14 which form a 3 base pair duplex stem without a base separation between the binding oligonucleotides on the target, showed comparable activity to that of the combination of oligonucleotides 12 and 16, which fom a 5 base pair duplex stem but with a one base separation. This result correlates well with the Tm daca (Table 3).
  • FIG. 8 shows the relationship between HIV-1 inhibition and Tm of the complex formed.
  • cooperative oligonucleotides of the invention can be directed to any target oligonucleotide.
  • Other non-limiting examples of such cooperative oligonucleotides include those specific for influenza nucleic acid targets which are shown below in Table 4.
  • sequence-specific and cooperative binding of short oligonucleotides that bind to adjacent sites are useful to target sequences with point mutations specifically.
  • undesirable non-sequence specific effects can be reduced by using at least two short oligonucleotides that can bind to a longer target sequence rather than one long oligonucleotide that binds to the same length of the target sequence.
  • long oligonucleotides that contain a modified backbone, such as phosphorothioate ⁇ activate complement, which have adverse cardiovascular effects (Galbraith et al. (1994) Antisense Res. Dev. 4:201-207; and Cornish et al. (1993) Pharmacol. Commun.
  • combination oligonucleotides represent an alternative therapeutic strategy to the use of a single oligonucleotide, in cases in which use of the latter is limited by concentration and chain length constraints, and the associated problems of toxicity and production costs.
  • the synthetic cooperative oligonucleotides of the invention also may be used to identify the presence of the nucleic acids of a particular virion or bacteria in cell cultures, for example, by labelling the oligonucleotide and screening for double-stranded, labelled DNA in the cells by in situ hybridization or some other art-recognized detection method.
  • the function of various genes in an animal including those essential to animal development can be examined using the cooperative oligonucleotides of the invention.
  • gene function can only be examined by the arduous task of making a "knock out" animal such as a mouse. This task is difficult, time-consuming and cannot be accomplished for genes essential to animal development since the "knock out” would produce a lethal phenotype.
  • the present invention overcomes the shortcomings of this model.
  • antisense oligonucleotides can bind to a target single-stranded nucleic acid molecule according to the Watson-Crick or the Hoogsteen rule of base pairing, and in doing so, disrupt the function of the target by one of several mechanisms: by preventing the binding of factors required for normal transcription, splicing, or translation; by triggering the enzymatic destruction of mRNA by RNase H if a contiguous region of deoxyribonucleotides exists in the oligonucleotide, an'-l or by destroying the target via reactive groups attached directly to the antisense oligonucleotide.
  • oligonucleotides are useful therapeutically by their ability to control or down- regulate the expression of a particular gene in a cell, e.g., in a cell culture or in an animal, according to the method of the present invention.
  • the cooperative oligonucleotides of the invention may also be used to inhibit transcription of any gene in a cell, including a foreign gene.
  • the cooperative oligonucleotides as provided by the invention may be use to inhibit the expression of HIV genes within infected host cells and thus to inhibit production of HIV virions by those cells .
  • the synthetic oligonucleotides of the invention are thus useful for treatment of HIV infection and AIDS in mammals, particularly the treatment of mammals used as animal models to study HIV infection and AIDS.
  • the synthetic oligonucleotides of the invention are also useful for treatment of humans infected with HIV and those suffering from AIDS.
  • the synthetic oligonucleotides of the invention may be used as a pharmaceutical composition when combined with a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable means a non-toxic material that does not interfere with the effectiveness of the biological activity of the active ingredient (s).
  • the characteristics of the carrier will depend on the route of administration.
  • Such a composition may contain, in addition to the synthetic oligonucleotide and carrier, diluents, fillers, salts, buffers, stabilizers, solubilizers, and other materials well known in the art.
  • the pharmaceutical composition of the invention may also contain other active factors and/or agents which enhance inhibition of virus or bacterial production by infected cells.
  • combinations of synthetic oligonucleotides may be used in the pharmaceutical compositions of the invention.
  • the pharmaceutical composition of the invention may further contain nucleotide analogs such as azidothymidine, dideoxycytidine, dideotylnosine, and the like.
  • nucleotide analogs such as azidothymidine, dideoxycytidine, dideotylnosine, and the like.
  • additional factors and/or agents may be included in the pharmaceutical composition to produce a synergistic effect with the synthetic oligonucleotide of the invention, or to minimize side-effects caused by the synthetic oligonucleotide of the invention.
  • the synthetic oligonucleotide of the invention may be included in formulations of a particular anti-HIV factor and/or agent to minimize side effects of the anti-HIV factor and/or agent.
  • the pharmaceutical composition of the invention may be in the form of a liposome in which the synthetic oligonucleotides of the invention is combined, in addition to other pharmaceutically acceptable carriers, with amphipathic agents such as lipids which exist in aggregated form as micelles, insoluble monolayers, liquid crystals, or lamellar layers which are in aqueous solution.
  • Suitable lipids for liposomal formulation include, without limitation, monoglycerides, diglycerides, sulfatides, lysolecithin, phospholipids, saponin, bile acids, and the like. Preparation of such liposomal formulations is within the level of skill in the art, as disclosed, for example, in U.S. Patent No. 4,235,871; U.S. Patent No. 4,501,728; U.S. Patent No. 4,837,028; and U.S. Patent No. 4,737,323.
  • the pharmaceutical composition of the invention may further include compounds which enhance delivery of oligonucleotides into cells, as described in WO 95/32739.
  • the term "therapeutically effective amount” means the total amount of each active component of the pharmaceutical composition or method that is sufficient to show a meaningful patient benefit, e.g., healing of chronic conditions characterized by HIV and associated infections and complications or by other viral infections or increase in rate of healing of such conditions.
  • a meaningful patient benefit e.g., healing of chronic conditions characterized by HIV and associated infections and complications or by other viral infections or increase in rate of healing of such conditions.
  • the term refers to that ingredient alone.
  • the term refers to combined amounts of the active ingredients that result in the therapeutic effect, whether administered in combination, serially or simultaneously.
  • a therapeutically effective amount of one or more of the synthetic oligonucleotide of the invention is administered to a mammal infected with HIV.
  • the synthetic oligonucleotide of the invention may be administered in accordance with the method of the invention either alone or in combination with other therapies such as treatments employing cytokines, lymphokines, other hematopoietic factors, other anti-viral agents, and the like.
  • the synthetic oligonucleotide of the invention may be administered either simultaneously with the cytokine(s), lymphokine (s), other hematopoietic factor (s), other antiviral agents, and the like, or sequentially. If administered sequentially, the attending physician will decide on the appropriate sequence of administering the synthetic oligonucleotide of the invention in combination with cytokine(s), lymphokine (s), other hematopoietic factor (s), anti-viral agents, and the like.
  • Administration of the synthetic oligonucleotide of the invention used in the pharmaceutical composition or to practice the method of the present invention can be carried out in a variety of conventional ways, such as oral ingestion, inhalation, or cutaneous, subcutaneous, or intravenous injection. Intravenous administration to the patient is preferred.
  • the synthetic oligonucleotide When a therapeutically effective amount of synthetic oligonucleotide of the invention is administered orally, the synthetic oligonucleotide will be in the form of a tablet, capsule, powder, solution or elixir.
  • the pharmaceutical composition of the invention may additionally contain a solid carrier such as a gelatin or an adjuvant.
  • the tablet, capsule, and powder contain from about 5 to 95% synthetic oligonucleotide and preferably from about 25 to 90% synthetic oligonucleotide.
  • a liquid carrier such as water, petroleum, oils of animal or plant origin such as peanut oil, mineral oil, soybean oil, sesame oil, or synthetic oils may be added.
  • the liquid form of the pharmaceutical composition may further contain physiological saline solution, dextrose or other saccharide solution, or glycols such as ethylene glycol, propylene glycol or polyethylene glycol.
  • the pharmaceutical composition contains from about 0.5 to 90% by weight of the synthetic oligonucleotide and preferably from about 1 to 50% synthetic oligonucleotide.
  • the synthetic oligonucleotide will preferably be in the form of a pyrogen-free, parenterally acceptable aqueous solution.
  • a preferred pharmaceutical composition for intravenous, cutaneous, or subcutaneous injection should contain, in addition to the synthetic oligonucleotide, an isotonic vehicle such as Sodium Chloride Injection, Ringer's Injection, Dextrose Injection, Dextrose and Sodium Chloride Injection, Lactated Ringer's Injection, or other vehicle as known in the art.
  • the pharmaceutical composition of the present invention may also contain stabilizers, preservatives, buffers, antioxidants, or other additives known to those of skill in the art.
  • the amount of synthetic oligonucleotide in the pharmaceutical composition of the present invention will depend upon the nature and severity of the condition being treated, and on the nature of prior treatments which the patient has undergone. Ultimately, the attending physician will decide the amount of synthetic oligonucleotide with which to treat each individual patient. Initially, the attending physician will administer low doses of the synthetic oligonucleotide and observe the patient's response. Larger doses of synthetic oligonucleotide may be administered until the optimal therapeutic effect is obtained for the patient, and at that point the dosage is not increased further. It is contemplated that the various pharmaceutical compositions used to practice the method of the present invention should contain about 1 ng to about 100 mg of synthetic oligonucleotide per kg body weight.
  • the duration of intravenous therapy using the pharmaceutical composition of the present invention will vary, depending on the severity of the disease being treated and the condition and potential idiosyncratic response of each individual patient. It is contemplated that the duration of each application of the synthetic oligonucleotide will be in the range of 12 to 24 hours of continuous intravenous administration. Ultimately, the attending physician will decide on the appropriate duration of intravenous therapy using the pharmaceutical composition of the present invention.
  • oligonucleotides were purified on reverse phase (C 18 ) HPLC, detritylated, desalted (Waters C 18 sep-pack cartridges (Waters, Milford, MA), and checked for purity by polyacrylamide gel electrophoresis (Manniatis et al. in Molecular Cloning (A Laboratory Manual) , Cold Spring Harbor Laboratory, Cold Spring Harbor, NY).
  • Cooperative oligoribonucleotides and hybrids are prepared according to the method(s) of Metelev et al. (FEBS. Lett. (1988) 226:232-234; and Atabekov et al. (1988) FEBS. Lett. 232:96-98.
  • Cooperative phosphorothioate oligonucleotides for RNase H and tissue culture experiments were synthesized as above but using sulfurizing agent as oxidant instead of normal iodine oxidant.
  • Post- synthetic processing was carried out exactly as above but desalting was performed by dialysis for 72 hours against double distilled water.
  • UV Melting Studies were carried out in 150 mM sodium chloride, 10 mM sodium dihydrogen phosphate, and 2 mM magnesium chloride, pH 7.4 buffer. The oligonucleotide concentration was 0.36 ⁇ M as single strand. The oligonucleotides were mixed in buffer, heated to 95°C, cooled down to room temperature, and left at 4°C overnight. Thermal denaturation profiles were recorded at 260 nm at a heating rate of 0.5°C/min on a spectrophotometer ( Perkin-Elmer Lamba2, (Norwalk CT) equipped with a peltier thermal controller and attached to a personal computer for data collection. The (Tm) melting temperatures were measured from first deri vative plots (dA/dT vs T). Each value is an average of two separate runs and the values are within ⁇ l.0°C range.
  • RNA target SEQ ID NO: 22
  • SEQ ID NO: 22 was labelled at its 3'-end using terminal transferase and [ ⁇ -
  • RNA End-labelled RNA (3000-5000 cpm) was incubated with 1 to 1.5 ratio of the oligonucleotides in 30 ⁇ l of 20 mM Tris-HCl, pH 7.5, 10 mM MgCl 2 , 10 mM KCl, 0.1 mM DTT, 5% sucrose (w/v), and 40 units of RNasin (Promega, Madison, WI) at 4°C overnight. An aliquot (7 ⁇ l) was taken out as control, 1 ⁇ l (0.8 unit) of E. coli RNase H (Promega, Madison, WI ) was added to the remaining reaction mixture and incubated at room temperature.
  • the effect of the antisense oligonucleotides on the replication of HIV-1 during an acute infection was determined.
  • the test system is a modification of the standard cytopathic effect (CPE) -based MT-2 cell assay (Posner et al. (1991) J. Immunol. 146:4325; Pawels et al. (1988) J. Virol. Methods 20:309; Mosmann (1983) J. Immunol. Methods 65:55).
  • CPE cytopathic effect

Landscapes

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

Abstract

L'invention concerne une composition comprenant au moins deux oligonucléotides coopératifs synthétiques, comportant chacun une région complémentaire d'une région de régions en tandem, ne se chevauchant pas, d'un acide nucléique simple-brin cible. Chaque nucléotide comprend en outre, à une extrémité, un domaine de dimérisation, les domaines de dimérisation des oligonucléotides étant complémentaires. L'invention concerne également des structures en duplex, des complexes ternaires, des formulations pharmaceutiques, ainsi que des procédés faisant appel aux oligonucléotides coopératifs selon l'invention.
PCT/US1997/005683 1995-04-12 1997-04-04 Oligonucleotides cooperatifs Ceased WO1997038097A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU27234/97A AU2723497A (en) 1996-04-04 1997-04-04 Cooperative oligonucleotides

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
US08/420,672 US6372427B1 (en) 1995-04-12 1995-04-12 Cooperative oligonucleotides
US62796796A 1996-04-04 1996-04-04
US08/627,967 1996-04-04

Publications (1)

Publication Number Publication Date
WO1997038097A1 true WO1997038097A1 (fr) 1997-10-16

Family

ID=27024956

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1997/005683 Ceased WO1997038097A1 (fr) 1995-04-12 1997-04-04 Oligonucleotides cooperatifs

Country Status (1)

Country Link
WO (1) WO1997038097A1 (fr)

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001051630A1 (fr) * 2000-01-07 2001-07-19 Baylor University Compositions antisens et methodes afferentes
US6518017B1 (en) 1997-10-02 2003-02-11 Oasis Biosciences Incorporated Combinatorial antisense library
EP1476564A4 (fr) * 2002-01-22 2006-03-15 Idera Pharmaceuticals Inc Oligonucleotides cooperatifs
US8153772B2 (en) 1997-10-02 2012-04-10 Gen-Probe Incorporated Oligonucleotide probes and primers comprising universal bases for diagnostic purposes

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0185494A2 (fr) * 1984-12-13 1986-06-25 Applied Biosystems, Inc. Détection de la séquence spécifique des acides nucléiques
GB2225112A (en) * 1988-11-22 1990-05-23 Ici Plc Hybridisation probes
WO1991006626A2 (fr) * 1989-10-23 1991-05-16 Gilead Sciences, Inc. Formation d'une triple chaine helicoidale en therapie oligonucleotidique
WO1994017086A1 (fr) * 1993-01-25 1994-08-04 Apollon, Inc. Regulation de genes par ciblage d'une helice triple intramoleculaire potentielle
WO1994023028A2 (fr) * 1993-03-31 1994-10-13 Hybridon, Inc. Oligonucleotides modifies ayant une activite antigrippe amelioree
WO1995001985A1 (fr) * 1993-07-09 1995-01-19 Lynx Therapeutics, Inc. Composes anti-sens d'oligonucleotides auto-liants ayant des applications en matiere de diagnostic et de therapie
WO1996032474A1 (fr) * 1995-04-12 1996-10-17 Hybridon, Inc. Oligonucleotides cooperants

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0185494A2 (fr) * 1984-12-13 1986-06-25 Applied Biosystems, Inc. Détection de la séquence spécifique des acides nucléiques
GB2225112A (en) * 1988-11-22 1990-05-23 Ici Plc Hybridisation probes
WO1991006626A2 (fr) * 1989-10-23 1991-05-16 Gilead Sciences, Inc. Formation d'une triple chaine helicoidale en therapie oligonucleotidique
WO1994017086A1 (fr) * 1993-01-25 1994-08-04 Apollon, Inc. Regulation de genes par ciblage d'une helice triple intramoleculaire potentielle
WO1994023028A2 (fr) * 1993-03-31 1994-10-13 Hybridon, Inc. Oligonucleotides modifies ayant une activite antigrippe amelioree
WO1995001985A1 (fr) * 1993-07-09 1995-01-19 Lynx Therapeutics, Inc. Composes anti-sens d'oligonucleotides auto-liants ayant des applications en matiere de diagnostic et de therapie
WO1996032474A1 (fr) * 1995-04-12 1996-10-17 Hybridon, Inc. Oligonucleotides cooperants

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
COLOCCI N ET AL: "COOPERATIVE BINDING OF 8-MER OLIGONUCLEOTIDES CONTAINING 5-(1-PROPYNYL)-2'-DEOXYURIDINE TO ADJACENT DNA SITES BY TRIPLE-HELIX FORMATION", JOURNAL OF THE AMERICAN CHEMICAL SOCIETY, vol. 116, 1994, pages 785/786, XP002018814 *
HERTEL K J ET AL: "NUMBERING SYSTEM FOR THE HAMMERHEAD", NUCLEIC ACIDS RESEARCH, vol. 20, no. 12, 25 June 1992 (1992-06-25), pages 3252, XP000278353 *
KANDIMALLA, EKAMBAR R. ET AL: "Design, biochemical, biophysical and biological properties of cooperative antisense oligonucleotides", NUCLEIC ACIDS RES. (09-1995), 23(17), 3578-84 CODEN: NARHAD;ISSN: 0305-1048, XP002037285 *

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6518017B1 (en) 1997-10-02 2003-02-11 Oasis Biosciences Incorporated Combinatorial antisense library
EP1019539A4 (fr) * 1997-10-02 2005-03-09 Gen Probe Inc Bibliotheque antisens combinatoire
US7790877B2 (en) 1997-10-02 2010-09-07 Gen-Probe, Incorporated Antisense oligonucleotides with increased RNase sensitivity
US8153772B2 (en) 1997-10-02 2012-04-10 Gen-Probe Incorporated Oligonucleotide probes and primers comprising universal bases for diagnostic purposes
WO2001051630A1 (fr) * 2000-01-07 2001-07-19 Baylor University Compositions antisens et methodes afferentes
EP1476564A4 (fr) * 2002-01-22 2006-03-15 Idera Pharmaceuticals Inc Oligonucleotides cooperatifs

Similar Documents

Publication Publication Date Title
US6372427B1 (en) Cooperative oligonucleotides
US5684147A (en) Therapeutic anti-HIV anti-viral oligonucleotides and pharmaceutical formulations thereof
US5968909A (en) Method of modulating gene expression with reduced immunostimulatory response
DK168061B1 (da) Med mrna hybridiserbart anti-viralt middel
EP0677056B1 (fr) Alkylphosphonates et alkylphosphonothioates d'oligonucleotides
JP5265067B2 (ja) 修飾ＣｐＧジヌクレオシドを有するオリゴヌクレオチドの使用方法
AU711792B2 (en) G cap-stabilized oligonucleotides
US10301628B2 (en) Treatment of idiopathic pulmonary fibrosis using RNA complexes that target connective tissue growth factor
AU3041701A (en) Therapeutic uses of lna-modified oligonucleotides
WO1995003406A2 (fr) Augmentation de l'inhibition oligonucleotidique de la production proteique, de la proliferation cellulaire et/ou de la multiplication d'agents pathogenes de maladie infectieuse
WO1995003406A9 (fr) Augmentation de l'inhibition oligonucleotidique de la production proteique, de la proliferation cellulaire et/ou de la multiplication d'agents pathogenes de maladie infectieuse
WO1997038097A1 (fr) Oligonucleotides cooperatifs
CA2211877A1 (fr) Inhibiteurs de la transcription du virus de l'immunodeficience humaine et procedes pour les utiliser
AU2003236659B2 (en) Cooperative oligonucleotides
WO2007084359A2 (fr) Compositions et procedes pour le traitement des infections grippales
AU2003236659A1 (en) Cooperative oligonucleotides
WO1998049287A2 (fr) Oligonucleotides antisens specifiques d'une thymidylate synthase
WO1999027087A1 (fr) Oligonucleotides anti-sens specifiques de la cdk4
EP1007098B1 (fr) Retro-regulation d'expression genique par administration colo-rectale d'oligonucleotides synthetiques
WO2024077262A2 (fr) Procédés et compositions pour le silençage de l'expression d'elavl2 pour le traitement d'une maladie
Galderisi et al. Antisense oligonucleotides as drugs for HIV treatment
CA2283626A1 (fr) Oligonucleotides modifies specifiques de la proteine kinase a et methodes d'utilisation
JPH0977670A (ja) 抗ウイルス剤

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

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

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): GH KE LS MW SD SZ UG AT BE CH DE DK ES FI FR GB GR IE IT LU MC NL PT SE BF BJ CF CG CI

121 Ep: the epo has been informed by wipo that ep was designated in this application
NENP Non-entry into the national phase

Ref country code: CA

REG Reference to national code

Ref country code: DE

Ref legal event code: 8642

NENP Non-entry into the national phase

Ref country code: JP

Ref document number: 97536388

Format of ref document f/p: F

122 Ep: pct application non-entry in european phase