EP1224280A2 - Conception d'un oligonucleotide de recrutement de rnase h a haute affinite - Google Patents

Conception d'un oligonucleotide de recrutement de rnase h a haute affinite

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Publication number
EP1224280A2
EP1224280A2 EP00962273A EP00962273A EP1224280A2 EP 1224280 A2 EP1224280 A2 EP 1224280A2 EP 00962273 A EP00962273 A EP 00962273A EP 00962273 A EP00962273 A EP 00962273A EP 1224280 A2 EP1224280 A2 EP 1224280A2
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EP
European Patent Office
Prior art keywords
lna
oxy
oligo
high affinity
monomers
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EP00962273A
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German (de)
English (en)
Inventor
Claes Wahlestedt
Mogens Havsteen Jakobsen
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Exiqon AS
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Exiqon AS
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Publication of EP1224280A2 publication Critical patent/EP1224280A2/fr
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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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
    • C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
    • C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
    • C07H19/06—Pyrimidine radicals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
    • C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
    • C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
    • C07H19/16—Purine radicals
    • 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
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/32—Chemical structure of the sugar
    • C12N2310/323—Chemical structure of the sugar modified ring structure
    • 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
    • C12N2310/34—Spatial arrangement of the modifications
    • C12N2310/341—Gapmers, i.e. of the type ===---===
    • 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
    • C12N2310/34—Spatial arrangement of the modifications
    • C12N2310/345—Spatial arrangement of the modifications having at least two different backbone modifications

Definitions

  • the present invention relates to the field of bicyclic DNA analogues which are useful for designing oligomers that forms high affinity duplexes with complementary RNA wherein said duplexes are substrates for RNase H.
  • the oligonucleotides may be partially or fully composed of bicyclic DNA analogues.
  • antisense relates to the use of oligonucleotides as therapeutic agents. Briefly, an antisense drug operates by binding to the mRNA thereby blocking or modulating its translation into protein. Thus, antisense drugs may be used to directly block the synthesis of disease causing proteins. It may, of course, equally well be used to block synthesis of normal proteins in cases where these participate in, and aggravate a pathophysiological process. Also, it ought to be emphasised that antisense drugs can be used to activate genes rather that suppressing them. As an example, this can be achieved by blocking the synthesis of a natural suppressor protein.
  • the hybridising oligonucleotide is thought to elicit its effect by either creating a physical block to the translation process or by recruiting a cellular enzyme (RNase H) that specifically degrades the mRNA part of the mRNA/antisense oligonucleotide duplex.
  • RNase H a cellular enzyme
  • oligonucleotides must satisfy a large number of different requirements to be useful as antisense drugs.
  • the antisense oligonucleotide must bind with high affinity and specificity to its target mRNA, must have the ability to recruit RNase H, must be able to reach its site of action within the cell, must be stable to extra - and intra- cellular nucleases both endo- and exo-nucleases, must be non-toxic/minimally immune stimulatory, etc.
  • the enzyme RNase H selectively binds to heterogeneous DNA/RNA duplexes and de- grades the RNA part of the duplex.
  • Homogeneous DNA/DNA and RNA/RNA duplexes which only differs molecularly from the DNA/RNA duplex at the 2 ' position (DNA/DNA: 2'- H/2 ' -H; RNA/RNA: 2 ' -OH/2 ' -OH and DNA/RNA: 2 ' -H/2 ' -OH) are not substrates for the enzyme. This suggests that either the molecular composition at the 2 ' position itself or the structural feature it imposes on the helix is vital for enzyme recognition. Consistent with this notion, all 2 ' -modified analogues that have so far been reported to exhibit increased affinity have lost the ability to recruit RNase H.
  • LNA Locked Nucleic Acid
  • oxy-LNA O-methylene
  • thio-LNA S-methylene
  • amino-LNA NH 2 -methylene moiety
  • oxy-LNA may be used in combination with non-oxy-LNA, such as standard DNA, RNA or other analogues, e.g. thio-LNA or amino- LNA to create high affinity, RNase H recruiting antisense compounds without the need to adhere to any fixed design.
  • non-oxy-LNA such as standard DNA, RNA or other analogues, e.g. thio-LNA or amino- LNA to create high affinity, RNase H recruiting antisense compounds without the need to adhere to any fixed design.
  • oxy-LNA monomer is defined herein as a nucleotide monomer of the formula la
  • X is oxygen;
  • B is a nucleobase;
  • R 1* , R 2 , R 3 , R 5 and R 5* are hydrogen;
  • P designates the radical position for an internucleoside linkage to a succeeding monomer, or a 5'- terminal group,
  • R 3* is an internucleoside linkage to a preceding monomer, or a 3'-terminal group;
  • R 2* and R 4* together designate -O-CH 2 - where the oxygen is attached in the 2'- position.
  • nucleobase covers the naturally occurring nucleobases adenine (A), guanine (G), cytosine (C), thymine (T) and uracil (U) as well as non-naturally occuring nucleo- bases such as xanthine, diaminopurine, 8-oxo-N 6 -methyladenine, 7-deazaxanthine, 7- deazaguanine, N 4 ,N 4 -ethanocytosin, N 6 ,N 6 -ethano-2,6-diaminopurine, 5-methylcytosine, 5-(C 3 -C 6 )-alkynylcytosine, 5-fluorouracil, 5-bromouracil, pseudoisocytosine, 2-hydroxy-5- methyl-4-triazolopyridin, isocytosine, isoguanin, inosine and the "non-naturally occurring" nucleobases described in Benner et al.
  • nucleobase thus includes not only the known purine and pyrimidine heterocycles, but also heterocyclic analogues and tautomers thereof. It should be clear to the person skilled in the art that various nucleobases which previously have been considered “non-naturally occurring” have subsequently been found in nature.
  • non-oxy-LNA monomer is broadly defined as any nucleoside (i.e. a glycoside of a het- erocyclic base) which is not itself an oxy-LNA but which can be used in combination with oxy-LNA monomers to construct oligos which have the ability to bind sequence specifically to complementary nucleic acids.
  • non-oxy-LNA monomers include 2'- deoxynucleotides (DNA) or nucleotides (RNA) or any analogues of these monomers which are not oxy-LNA, such as for example the thio-LNA and amino-LNA described by Wengel and coworkers (Singh et al. J. Org. Chem. 1998, 6, 6078-9, and the derivatives described in Susan M. Freier and Karl-Heinz Altmann, Nucleic Acids Research, 1997, vol 25, pp 4429-4443.
  • non-oxy-LNA monomer(s) into an oxy- LNA oligo may change the RNAseH recruiting characteristics of the oxy-LNA/non-oxy- LNA chimeric oligo.
  • the chimera may have an increased, unaltered or decreased ability to recruit RNAsdeH as compared to the corresponding all oxy-LNA oligo.
  • R 3* is a group P* which designates an internucleoside linkage to a preceding monomer, or a 3'-terminal group;
  • 6 -alkyl-aminocarbonyl mono- and di(C 1-6 -alkyl)amino-C 1 . 6 -alkyl-arninocarbonyl, d. 6 -alkyl-carbonylamino, carbamido, C ⁇ -6 -alkanoyloxy, sulphono, C 1-6 -alkylsulphonyloxy, nitro, azido, sul- phanyl, C 1-6 -alkylthio, halogen, photochemically active groups, thermochemically active groups, chelating groups, reporter groups, and ligands, said possible pair of non-geminal substituents thereby forming a monocyclic entity together with (i) the atoms to which said non-geminal substituents are bound and (ii) any intervening atoms; and
  • each of the substituents R 2 , R 2* , R 3 , R 4* which are present and not involved in the possible biradical is independently selected from hydrogen, optionally substituted C 1-6 -alkyl, optionally substituted C 2-6 -alkenyl, hydroxy, d. 6 -alkoxy, C 2 . 6 -alkenyloxy, carboxy, d-e- alkoxycarbonyl, C ⁇ _ 6 -alkylcarbonyl, formyl, amino, mono- and di(C 1 .
  • the monomer is not oxy-LNA.
  • non-oxy-LNA monomers are 2'-deoxyribonucleotides, ribonucleo- tides, and analogues thereof that are modified at the 2'-position in the ribose, such as 2 ' - O-methyl, 2 ' -fluoro, 2'-thfluoromethyl, 2 ' -O-(2-methoxyethyl), 2'-O-aminopropyl, 2 ' -O- dimethylamino-oxyethyl, 2 ' -O-fluoroethyl or 2'-O-propenyl, and analogues wherein the modification involves both the 2 ' and 3" position, preferably such analogues wherein the modifications links the 2'- and 3'-position in the ribose, such as those described by Wen- gel and coworkers (Nielsen et al., J.
  • non-oxy-LNA monomers having the ⁇ -D-ribo configuration are often the most applicable, further interesting examples (and in fact also applicable) of non-oxy-LNA are the stereoi- someric of the natural ⁇ -D-ribo configuation.
  • ⁇ -L-ribo particularly interesting are the ⁇ -L-ribo, the ⁇ - D-xylo and the ⁇ -L-xylo configurations (see Beier et al., Science, 1999, 283, 699 and Es- chenmoser, Science, 1999, 284, 2118), in particular those having a 2'-4' -CH 2 -S-, -CH 2 - NH-, -CH 2 -O- or -CH 2 -NMe- bridge (see Wengel and coworkers in Rajwanshi et al., Chem. Commun., 1999, 1395 and Rajwanshi et al., Chem. Commun., 1999, submitted)
  • oligonucleotide which is the same as “oligomer” which is the same as “oligo” means a successive chain of nucleoside monomers (i.e. glycosides of heterocyclic bases) connected via internucleoside linkages.
  • 6 -alkyl and phenyl are especially preferred. Further illustrative examples are given in Mesmaeker et. al., Current Opinion in Structural Biology 1995, 5, 343-355 and Susan M. Freier and Karl-Heinz Altmann, Nucleic Acids Research, 1997, vol 25, pp 4429- 4443.
  • the left-hand side of the internucleoside linkage is bound to the 5-membered ring as substituent P * at the 3'-position, whereas the right-hand side is bound to the 5'-position of a preceding monomer.
  • the term "succeeding monomer” relates to the neighbouring monomer in the 5'-terminal direction and the “preceding monomer” relates to the neighbouring monomer in the 3'- terminal direction.
  • Monomers are referred to as being "complementary” if they contain nucleobases that can form hydrogen bonds according to Watson-Crick base-pairing rules (e.g. G with C, A with T or A with U) or other hydrogen bonding motifs such as for example diaminopurine with T, inosine with C, pseudoisocytosine with G, etc.
  • Watson-Crick base-pairing rules e.g. G with C, A with T or A with U
  • other hydrogen bonding motifs such as for example diaminopurine with T, inosine with C, pseudoisocytosine with G, etc.
  • modified oxy-LNA oligo contain at least two non-oxy-LNA monomers these may contain the same or different nucleobases at the 1 '-position and be identical at all other positions or they may contain the same or different nucleobases at the 1 '-position and be non-identical at at least one other position.
  • the present invention describes a method for degrading RNA in-vivo (in a cell or organism) or in-vitro by providing a high affinity oligonucleotide which activates RNaseH when the high affinity oligonucleotide is hybridised to a complementary RNA target sequence
  • said high affinity oligonucleotide may consist of oxy-LNA monomers exclusively.
  • the high affinity oligonucleotide may also consist of both oxy-LNA and non- oxy-LNA monomers, in this case the high affinity oligonucleotide contains at the most five, e.g. 4, e.g. 3 , e.g.
  • said high affinity oligonucleotide consists of both oxy-LNA and non-oxy- LNA monomers, wherein none of the non-oxy-LNA monomers are located adjacent to each other.
  • the high affinity oligonucleotide may also contain one or more segments of contigous non-oxy-LNA monomers. For instance, a stretch of contigous non-oxy-LNA monomers may be located in the centre of the oligonucleotide and with flanking segments consisting of oxy-LNA monomers. Alternatively the stretch of contigous non-oxy-LNA monomers may be located at either or both ends. Also, the oxy-LNA segement(s) may be either contigous or interrupted by 1 or more non-oxy-LNA monomers. Also, the high affinity oligonucleotide may comprise more than one type of internucleoside linkage such as for example mixes of phosphordiester and phosphorothioate linkages.
  • the resulting high affinity oligo containing oxy-LNA monomers and/or non-oxy-LNA monomers can thus be characterized by the general formula
  • X is oxy-LNA and Y is non-oxy-LNA, wherein m and p are integers from 0 to 30, n is an integer from 0 to 3 and q is an integer from 1 to 10 with the proviso that the sum of m+n+p multiplied with q is in the range of 6-100, such as 8, e.g. 9, e.g. 10, e.g. 11 , e.g. 12, e.g. 13, e.g. 14, e.g. 15, e.g. 16, e.g. 17, e.g. 18, e.g. 19, e.g. 20, e.g. 21 , e.g. 22, e.g. 23, e.g. 24, e.g.
  • the present invention provides oligos which combine high affinity and specificity for their target RNA molecules with the ability to recruit RNAseH to an extend that makes them useful as antisense therapeutic agents.
  • the oligos may be composed entirely of oxy-LNA monomers or they may be composed of both oxy and non-oxy-LNA monomers.
  • the RNAseH recruiting characteristics of the chimeric oligo may be similar to, or different from, that of the corresponding oxy-LNA oligo.
  • non-oxy-LNA monomer(s) is/are used in such a way that they do not change the RNAseHspanninging characteristics of the oxy-LNA/non-oxy-LNA chimeric oligo compared to the corresponding all oxy-LNA oligo.
  • the non-oxy-LNA monomer(s) is/are used purposely to change the RNAseH recruiting characteristics of an oxy-LNA oligo, either increasing or decreasing its efficiency to promote RNAseH cleavage when hybridised to its complementary RNA target compared to the corresponding all oxy-LNA oligo.
  • the ability of the chimeric oligo to discriminate between its complementary target RNA and target RNAs containing one or more Watson-Crick mismatches may be different from the ability of the corresponding all oxy-LNA oligo to discriminate between its matched and mismatched target RNAs.
  • an oxy-LNA oligo to discriminate be- tween a complementary target RNA and a single base mismatched target RNA can be enhanced by incorporating non-oxy-LNA monomer(s), such as for instance DNA, RNA, thio-LNA or amino-LNA, either at, or close to, the mismatched position as described in applicant's Danish patent application entitled “Metod of increasing the specificity of oxy- LNA oligonuclotides" filed on the same day as the present application.
  • non-oxy-LNA monomer(s) such as for instance DNA, RNA, thio-LNA or amino-LNA
  • non-oxy-LNA monomer(s) is/are used purposely to construct an oxy-LNA/non-oxy-LNA oligo which exhibit increased specificity but unaltered RNAseH recruiting characteristics compared to the corresponding all oxy-LNA oligo.
  • the non-oxy-LNA monomer(s) is/are used purposely to construct an oxy-LNA/non-oxy-LNA oligo which exhibit both increased specificity and altered RNAseH recruiting characteristics compared to the corresponding all oxy-LNA oligo
  • oligonucleotide of the present invention may be conjugated with compounds selected from proteins, amplicons, enzymes, polysaccharides, antibodies, nap- tens and peptides. Examples
  • Example 1 LNA containing oligonucleotides recruit RNase H
  • DNA control 5'- gtgtccgagacgttg-3' phosphorothioate control; 5' -gtgtccgagacgttg-3'
  • LNA qab-mer 5'-GTGTccgagaCGTTG-3' (LNA in capital letters, DNA is small letters) and LNA-mix-mer; 5'-gTgTCCgAgACgTTg-3' (LNA in capital letters, DNA is small letters)
  • the promoter sequence for T7 polymerase recognition and initiation of transcription were contained, followed by the DNA sequence coding for the target-RNA sequence.
  • the two complementary oligonucleotides were heated to 80°C for 10min to produce the linearised double-strand template.
  • a 20 ⁇ l in vitro transcription reaction containing 500 ⁇ M each of ATP, GTP and CTP, 12 ⁇ M of UTP, ap- prox.
  • RNA polymerase 50 ⁇ Ci of ⁇ - 32 P UTP, 1 x transcription buffer (Tris-HCI, pH 7.5), 10mM dithiotretiol, 1% BSA, 20 U of RNasin ribonuclease inhibitor, 0.2 ⁇ l template and 250 units T7 RNA polymerase.
  • the inclusion of RNasin inhibitor was to prevent degradation of the target- RNA from ribonucleases. The reactions were carried out at 37°C for 2h to produce the desired 24mer 32 U-labelled RNA run-off transcript.
  • RNA sequence was then purified via ethanol precipitation, the supematants filtered through a Millipore (0.45m) and collected by ethanol precipitation. The pellets were diluted in TE-buffer and subsequently subjected to RNAse H digestion assay.
  • the decrease of intact substrate i.e.
  • the 24-mer - 32 P UTP labelled target RNA sequence was assayed over time as follows.
  • the reactions were carried out in a total volume of 110 ⁇ l and contained (added in the order mentioned): 1 x nuclease-free buffer (20mM Tris-HCI, pH 7.5, 40mM KCI, 8mM MgCI 2 , 0.03 mg/ml BSA), 10mM dithiotretiol, 4% glycerol, 100nM of oligonucleotide, 3 Units RNasin inhibitor, labelled target RNA strand and 0.1 U of RNase H. An excess of oligonucleotide was added to each reaction to ensure full hybridisation of the RNA target sequences.
  • Two negative controls were also included and were prepared as above but (1) without any oligonucleotide, or (2) without RNase H added to the reaction mixture. All the reactions were incubated at 37°C. At time points 0, 10, 20, 40 and 60 min., 10 ⁇ l aliquots were taken and immediately added to ice-cold formamide loading buffer to quench the reaction and stored at -20°C. The samples were heated to 85°C for 5 min. prior to loading and running on a 15% polyacrylamide gel containing 7M urea. The gels were vacuum dried and exposed to autoradiographic films over night and subsequently subjected to densitometric calculations using the Easy Win imaging software (Hero Labs). The volume density of intact target RNA were calculated in each lane with correction for background. The volume density for the time zero sample was set as reference value for each incubation. Relative values for the other time-points samples in the corresponding incubation were calculated based on these reference values.
  • FIG. 1 shows the results of the RNase H experiments.
  • the control DNA and phosphorothioate oligonucleotides both recruit RNAse H very efficiently.
  • the LNA oligonucleotide which contains a contiguous stretch of six DNA monomers in the middle recruits RNAse H efficiently.
  • the LNA mix-mer which contains only single DNA monomers interdispersed between LNA mono- mers also recruits RNAse H.
  • the activity of RNase H is not contingent on a contiguous stretch of DNA or phosphorothioated bases when LNA is used as a component of the oligonucleotide.

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Abstract

La présente invention a trait au domaine des analogues d'ADN bicycliques, par exemple les ALN et les modifications d'ALN, qui sont utilisés pour mettre au point des oligomères formant des duplex à haute affinité avec les ARN complémentaires, les duplex étant des substrats de Rnase H. Les oligonucléotides de l'invention peuvent être partiellement ou entièrement composés d'analogues d'ALN possédant une très haute affinité et une très haute capacité de recrutement de la RNase H. Grâce à la présente invention, on peut utiliser l'oxy-ALN seul pour construire de nouvelles molécules antisens présentant une activité biologique améliorée. Dans un autre mode de réalisation, on peut utiliser l'oxy-ALN en combinaison avec des ALN non oxy, tels que l'ADN, l'ARN ordinaires ou analogues, par exemple, le thio-ALN ou l'amino-ALN, pour créer des composés antisens de recrutement de RNase H à haute affinité sans qu'il soit nécessaire de s'en tenir à une structure rigide.
EP00962273A 1999-10-04 2000-10-03 Conception d'un oligonucleotide de recrutement de rnase h a haute affinite Withdrawn EP1224280A2 (fr)

Applications Claiming Priority (5)

Application Number Priority Date Filing Date Title
DKPA199901422 1999-10-04
DK142299 1999-10-04
US15772499P 1999-10-05 1999-10-05
US157724P 1999-10-05
PCT/DK2000/000550 WO2001025248A2 (fr) 1999-10-04 2000-10-03 Conception d'un oligonucleotide de recrutement de rnase h a haute affinite

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EP1224280A2 true EP1224280A2 (fr) 2002-07-24

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JP (1) JP2003511016A (fr)
AU (1) AU7406700A (fr)
CA (1) CA2385853A1 (fr)
IL (1) IL148916A0 (fr)
WO (1) WO2001025248A2 (fr)

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WO2001025248A2 (fr) 2001-04-12
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WO2001025248A3 (fr) 2001-08-30
CA2385853A1 (fr) 2001-04-12

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