WO1994013789A2 - Oligonucleotides presentant une activite de clivage de l'arn - Google Patents

Oligonucleotides presentant une activite de clivage de l'arn Download PDF

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Publication number
WO1994013789A2
WO1994013789A2 PCT/GB1993/002486 GB9302486W WO9413789A2 WO 1994013789 A2 WO1994013789 A2 WO 1994013789A2 GB 9302486 W GB9302486 W GB 9302486W WO 9413789 A2 WO9413789 A2 WO 9413789A2
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WO
WIPO (PCT)
Prior art keywords
substituted
oligonucleotide
diol
formula
oligonucleotides
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/GB1993/002486
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English (en)
Other versions
WO1994013789A3 (fr
Inventor
Janos Ludwig
Fritz Benseler
Gerd Kotzorek
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.)
RIBONETICS GmbH
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RIBONETICS GmbH
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
Application filed by RIBONETICS GmbH filed Critical RIBONETICS GmbH
Priority to AU59741/94A priority Critical patent/AU5974194A/en
Priority to EP94905771A priority patent/EP0672122A1/fr
Publication of WO1994013789A2 publication Critical patent/WO1994013789A2/fr
Publication of WO1994013789A3 publication Critical patent/WO1994013789A3/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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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
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • 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
    • C12N2310/11—Antisense
    • C12N2310/111—Antisense spanning the whole gene, or a large part of it
    • 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
    • C12N2310/318—Chemical structure of the backbone where the PO2 is completely replaced, e.g. MMI or formacetal
    • C12N2310/3183—Diol linkers, e.g. glycols or propanediols
    • 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
    • C12N2310/33—Chemical structure of the base

Definitions

  • the present invention relates to oligonucleotides with RNA cleavage activity.
  • RNA is known to have endoribonuclease activity
  • Catalytic RNA molecules called ribozymes have been used to bind to target RNA molecules and catalyse their cleavage, thus blocking tne activity of the target RNA.
  • the so-called hammerhead ribozymes have been widely studied.
  • the recognition site and catalytic site of these oligoribonucleotides are well characterised and ricozymes containing a recognition sequence specific for any desired target RNA which contains a specified triplet can be constructed. These compounds can therefore ne considered as potential therapeutic agents with possibly higher biological activity than the simple antisense oligonucleotides (1) .
  • FIG. 1 The structure of a typical hammerhead ribozyme is shown in Figure 1.
  • a G C and U are all ribonucleotides.
  • the hammernead ribozyme has been shown to have no effect in prokaryotic cells wnen the ribozyme and target RNA were generated from different genes, whereas it can function at a 1:1 ribozyme target ratio if co-localization in the same cell is maintained.
  • Studies (1) have shown that a high (1000:1) ribozyme:sunstrate ratio is needed for inhibition in vivo in eukaryot ic cells . This suggests that the catalytic potential of the ribozyme is not being achieved.
  • Eckstein et al introduced 2'-ammo or 2'-F substituents into the pyrimidine positions of the hammerhead ribozyme (2) (3) .
  • the cleavage positions of the rioozymes in cellular extracts were not determined but the all-pyrimidme substitutions together with phosphorothiolate substitutions at the 3' terminus gave compounds with a markedly increased stability. The influence of these chemical modifications on the catalytic activity of the ribozymes was negligible.
  • Cedergreen et al found that mixed DNA / RNA oligomers with 4-7 noopositions are active in cleaving substrate RNA. These oligomers are three orders of magnitude more stable than the all -RNA ribozymes in incubation with RNase A and yeast extract.
  • McLaughlin et al (10 ) proposed a role for the 2'-OH as of G 5 or G 8 . They interact with H 2 O molecules bound in the first co-ordination sphere of the Mg 2+ cofactor.
  • the present invention provides in a first aspect oligonucleotides having the following sequence 3' 5 '
  • A is deoxyadenosine
  • G is deoxvguanosine
  • a is 2' substituted deoxyadenosine according to formula la below, or a 2' substituted arabinofuranosyl adenosine according to formula Ic below, or adenosine g is 2' substituted deoxvguanosine according to formula lb below or a 2' substituted arabinofuranosyl guanosine according to formula Id below, or guanosine
  • R 1 being -H, -C(O)OH, or -CH 2 -C(O)OH
  • oligonucleotide is other than adenosine or guanosine respectively;
  • P is either a) A-U or b) C-G
  • W is either i) nucleotide loop sequence IIa if P is A-U;
  • each diol bridge (Z) is of formula IV
  • n 1-10
  • n may be independently selected for each bridge from l to 10.
  • n is 3 in each bridge and there are four bridges;
  • m is 4.
  • X and Y are target specific recognition sequences made up of any deoxyribonucleosides N depending on the target RNA sequence.
  • X and Y may be of the same or different length. There is no need for the molecule to be symmetrical. Each may be 4 to 25 nucleotides long, preferably 6 to 20 nucleotides. If X and Y are too short the oligonucleotide looses its specificity.
  • X or Y may include stabilising modifications.
  • two or three natural 3' -5' phosphodiester linkages at at least the 3' end of X may be modified in an attempt to protect the oligonucleotide from attack by 3'-exonucieases.
  • 3'-5' phosphodiester linkage may be replaced by phosphorothiat-e linkages such as thiophosphodiester linkages.
  • the 2'-R substituent is a non-nucleophylic group which is isoteric and isopolar with the replaced -OH group.
  • the substituent has both H-bond donor and acceptor abilities.
  • a 2'-COOH substituent, which can chelate with Mg 2+ is advantageous because it is known to be important for Mg 2+ binding.
  • modifications according to the invention which involve the 2'-R substitution of nucleotides in the catalytic/cleavage region of the oligonucleotide can provide a desirable increase in stability against degradation and can increase catalytic activity due to improved Mg 2+ binding.
  • Oligonucleotides according to the invention with diol bridges can ne made by machine more easily and more cheaply than conventional ribozymes.
  • the oligonucleotides of the present invention may be used as intermediates for further modification to improve their ease of up- take by the cell (in comparison to unmodified oligonucleotides of the invention and known ribozymes), for example by the attachment of carrier molecules.
  • the emoodiments navmg diol bridges are considered to be particularly useful as intermediates for this purpose.
  • the oligonucleotides of the present invention are potential antagonists of a wide range of therapeutic targets which involve over-expression of products. By binding to specific targets on mRNA and cleaving the mRNA they can stop translation and hence "switch off" a specific gene. In cases where expression of a product (e.g. an enzyme or a protein) by a gene is causative of an illness or disfunction this could lead to a cure. Alternatively a putative gene as a source of a problem phenomenon could be "switched off” selectively and the tnerapeutic effect observed.
  • the oligonucleotides of the present invention are potential anticancer and antiviral agents and could also be used as anti-inflammatory and anti-ulcer drugs.
  • oligonucleotides of the present invention suggest that they could be used in nanomolar amounts. This offers a significant improvement over known ribozymes which have to be used in relatively large amounts to compensate for tneir mtracellular degradation by nucleases.
  • the oligonucleotides of the invention may achieve true catalytic activity: i.e. they will not be destroyed in the cleavage reaction.
  • the invention can also provide novel intermediates which are 2' substituted nucleosides needed to construct the oligonucleotides of the present invention.
  • Such reactive intermediates may be protected where appropriate by a removable protecting group.
  • the invention can also provide a method which comprises the synthesis of an oligonucleotides according to the invention from building blocks including deoxynucleotides and 2' substituted nucleosides.
  • Figure 1 shows the nucleotide sequence of a hammerhead ribozyme.
  • Figure 2 shows an example of therapeutic target interaction between the myc oncogene mRNA and an oligonucleotide of the invention of the type described in Example 1.
  • Figure 3 shows a synthetic scheme for producing 2' - CF 2 H substituted nucleosides.
  • Figure 4 shows a synthetic scheme for producing 2' - COOH substituted nucleosides.
  • Figure 5 shows a synthetic scheme for producing the nucleoside 9-(2'-C-difluoromethyl - ⁇ - D arabino - furanosyl) - 6 Ethoxy purine.
  • N represents any deoxyribonucleotide recognition sequence specific for the target RNA
  • N.N represents a thiophosphodiester linkage replacing a natural
  • A is deoxyadenosine
  • G is deoxyguanosine
  • a is 2' substituted deoxyadenosine according to formula la below
  • g is 2' substituted deoxyguanosine according to formula lb below
  • R 1 being -H, -C(O)OH or -CH 2 -C(O)OH and W is nucleotide loop sequence Ila.
  • N represents any deoxyribonucleotide recognition sequence specific for the target RNA
  • N.N represents a thiophosphodiester linkage replacing a natural
  • A is deoxyadenosine
  • G is deoxyguanosine
  • a is 2' substituted deoxyadenosine according to formula la below
  • g is 2' substituted deoxyguanosine according to formula lb below in which R is selected from
  • R 1 being -H, -C(O)OH or -CH 2 -C(O)OH
  • W is nucleotide loop sequence IIb.
  • N, N.N, A, C, G, U, a and g are as defined for Example 1 and the diol bridges are connected with phosphodiester or substituted neutral ohosohotriester derivative linkages.
  • N, N.N, A, C, G, U, a and g are as defined for Example 1 and the diol bridges are connected with phosphodiester or substituted neutral phosphotriester derivative linkages.
  • FIG. 2 A therapeutic target interaction between mRNA and an oligonucleotide of the invention is shown in Figure 2.
  • the -myc oncogene mRNA secondary structure is shown from nucleotide 1 to nucleotide 900. Translation starts at nucleotide 421 and the triplet cleavage site is positions 433 to 435 (GUU).
  • 3 is a purine or pyrimidine heterocycle, i.e. adenine, cytosme, guanme or uracil which may be protected where appropriate.
  • 6 -Ethoxypurme is a synthetic synthon for adenine; we did not convert to an adenosine derivative on the nucleoside level, because the 6-ethoxy group is a suitable base protecting group during oligonucleotide synthesis.

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

Abstract

L'invention concerne des oligonucléotides présentant une activité de clivage de l'ARN représentés par la séquence (I) dans laquelle, ----X et Y---- sont des séquences de reconnaissance de l'ARN spécifique à une cible, A, C, G et U sont des désoxynucléotides et au moins un a et/ou g représente respectivement un dérivé de guanosine ou d'adénosine à substitution 2', P correspond à a) A-U ou b) C-G et, W représente soit i) une séquence nucléotidique à boucle, ou ii) des ponts de diol connectés à des liaisons de dérivés de phosphodiester ou de phosphotriester neutre substitué. Lesdits oligonucléotides peuvent présenter une stabilité accrue contre la dégradation par RNase, une stabilité accrue contre la dégradation par exonucléase, ainsi qu'une plus grande facilité de fixation cellulaire.
PCT/GB1993/002486 1992-12-04 1993-12-03 Oligonucleotides presentant une activite de clivage de l'arn Ceased WO1994013789A2 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
AU59741/94A AU5974194A (en) 1992-12-04 1993-12-03 Oligonucleotides with rna cleavage activity
EP94905771A EP0672122A1 (fr) 1992-12-04 1993-12-03 Oligonucleotides presentant une activite de clivage de l'arn

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
GB929225427A GB9225427D0 (en) 1992-12-04 1992-12-04 Oligonucleotides with rna cleavage activity
GB9225427.5 1992-12-04

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WO1994013789A2 true WO1994013789A2 (fr) 1994-06-23
WO1994013789A3 WO1994013789A3 (fr) 1994-08-04

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EP (1) EP0672122A1 (fr)
AU (1) AU5974194A (fr)
GB (1) GB9225427D0 (fr)
WO (1) WO1994013789A2 (fr)

Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995006731A3 (fr) * 1993-09-02 1995-07-06 Ribozyme Pharm Inc Acide nucleique enzymatique contenant un non-nucleotide
WO1995006764A3 (fr) * 1993-09-03 1995-10-19 Ribonetics Gmbh Oligonucleotides ayant une activite de segmentation de l'arn
WO1997018312A1 (fr) * 1995-11-14 1997-05-22 Vimrx Holdings, Ltd. Oligomeres chimeriques possedant une activite de clivage de l'arn
EP0767657A4 (fr) * 1994-06-22 1999-01-20 Nexstar Pharmaceuticals Inc Nouveau procede de production de nucleosides connus et nouveaux, modifies en position 2' par deplacement nucleophile intramoleculaire
US6610478B1 (en) 1996-08-16 2003-08-26 Yale University Phenotypic conversion of cells mediated by external guide sequences
WO2003093290A3 (fr) * 2002-05-06 2004-03-18 Genelabs Tech Inc Derives nucleosidiques destines au traitement de l'infection par le virus de l'hepatite c
US6737236B1 (en) 1997-01-08 2004-05-18 Proligo, Llc Bioconjugation of macromolecules
US7098326B2 (en) 2002-01-23 2006-08-29 Sigma-Aldrich Co. Methods for the integrated synthesis and purification of oligonucleotides
US7427678B2 (en) 1998-01-08 2008-09-23 Sigma-Aldrich Co. Method for immobilizing oligonucleotides employing the cycloaddition bioconjugation method
US7615629B2 (en) 2002-12-31 2009-11-10 Sigma-Aldrich Co. Methods and compositions for the tandem synthesis of two or more oligonucleotides on the same solid support

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU730934B2 (en) * 1993-09-02 2001-03-22 Ribozyme Pharmaceuticals, Inc. Non-nucleotide containing enzymatic nucleic acid

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1991019789A1 (fr) * 1990-06-19 1991-12-26 Commonwealth Scientific And Industrial Research Organisation Endonucleases
AU649074B2 (en) * 1990-10-12 1994-05-12 Max-Planck-Gesellschaft Zur Forderung Der Wissenschaften E.V. Modified ribozymes

Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995006731A3 (fr) * 1993-09-02 1995-07-06 Ribozyme Pharm Inc Acide nucleique enzymatique contenant un non-nucleotide
WO1995006764A3 (fr) * 1993-09-03 1995-10-19 Ribonetics Gmbh Oligonucleotides ayant une activite de segmentation de l'arn
EP0767657A4 (fr) * 1994-06-22 1999-01-20 Nexstar Pharmaceuticals Inc Nouveau procede de production de nucleosides connus et nouveaux, modifies en position 2' par deplacement nucleophile intramoleculaire
US6090932A (en) * 1994-06-22 2000-07-18 Proligo Llc Method of preparation of known and novel 2'-modified nucleosides by intramolecular nucleophilic displacement
WO1997018312A1 (fr) * 1995-11-14 1997-05-22 Vimrx Holdings, Ltd. Oligomeres chimeriques possedant une activite de clivage de l'arn
US6610478B1 (en) 1996-08-16 2003-08-26 Yale University Phenotypic conversion of cells mediated by external guide sequences
US6737236B1 (en) 1997-01-08 2004-05-18 Proligo, Llc Bioconjugation of macromolecules
US7427678B2 (en) 1998-01-08 2008-09-23 Sigma-Aldrich Co. Method for immobilizing oligonucleotides employing the cycloaddition bioconjugation method
US7098326B2 (en) 2002-01-23 2006-08-29 Sigma-Aldrich Co. Methods for the integrated synthesis and purification of oligonucleotides
WO2003093290A3 (fr) * 2002-05-06 2004-03-18 Genelabs Tech Inc Derives nucleosidiques destines au traitement de l'infection par le virus de l'hepatite c
US7615629B2 (en) 2002-12-31 2009-11-10 Sigma-Aldrich Co. Methods and compositions for the tandem synthesis of two or more oligonucleotides on the same solid support

Also Published As

Publication number Publication date
AU5974194A (en) 1994-07-04
EP0672122A1 (fr) 1995-09-20
GB9225427D0 (en) 1993-01-27
WO1994013789A3 (fr) 1994-08-04

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