WO1994012633A1 - Oligonucleotides stables - Google Patents

Oligonucleotides stables Download PDF

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Publication number
WO1994012633A1
WO1994012633A1 PCT/GB1993/002409 GB9302409W WO9412633A1 WO 1994012633 A1 WO1994012633 A1 WO 1994012633A1 GB 9302409 W GB9302409 W GB 9302409W WO 9412633 A1 WO9412633 A1 WO 9412633A1
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Prior art keywords
oligodeoxynucleotide
sequence
antisense
nucleotide
hairpin loop
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PCT/GB1993/002409
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English (en)
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Ilyas Mahmood Khan
Judy Margaret Coulson
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Stiefel Laboratories, Inc.
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Priority to AU55686/94A priority Critical patent/AU5568694A/en
Publication of WO1994012633A1 publication Critical patent/WO1994012633A1/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
    • 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
    • C12N2310/50—Physical structure
    • C12N2310/53—Physical structure partially self-complementary or closed

Definitions

  • the invention relates to oligonucleotides and in particular to oligodeoxynucleotides which have been modified so that they are relatively resistant to degradation by nucleases.
  • the invention has particular relevance to antisense oligodeoxynucleotides and the inhibition of gene expression both in vivo and in vitro.
  • Many disease states are the result of the expression of abnormal genes within cells. These may be human genes found mutated in many diverse premalignant and malignant lesions such as p53 which is a tumour suppressor gene. Foreign genes can become integrated into cellular DNA, for example after viral infection, and can subsequently be expressed by the cells. The expression of such genes provides obvious thera ⁇ Chamberic targets, and over the past years the idea of using antisense oligonucleotides as agents to specifically inhibit expression of these genes has become popular. Other viral infections, where no integration of viral DNA occurs are also suitable targets, as the translation of essential viral genes in infected cells may be blocked. Other disease states may also present opportunities for the therapeutic use of anti ⁇ sense oligonucleotides, as they result from over expression of normal gene products.
  • the basic principle of antisense technology relies on the complementary base pairing of a short oligonucleotide of either RNA or DNA, with DNA or with RNA transcribed from the gene of interest. This pairing can prevent the protein product of the gene from being expressed.
  • the base pairing can be chosen so that the antisense strand is complementary to a particular gene transcript.
  • Antisense mediated arrest of translation was first demonstrated in cell-free systems several years ago (Paterson, B. . et al. (Proc. Natl. Acad. Sci. USA 74:4370-4374 (1977)); Hastie, N.D. and Held, W.A. (Proc. Natl. Acad. Sci. USA 74:1257-1271 (1978)); and later in eukaryotic cells (Izant, J.G. and eintraub, H. (Cell 36: 1007-1015 (1984)). The potential of the technique was not widely investigated until the automated chemical synthesis of oligonucleotides became possible.
  • Antisense oligo ⁇ nucleotides have been shown to be capable of modifying the expression of their target genes in a wide range of experi ⁇ mental systems.
  • rabbit reticulocyte lysate (Minshull, J. and Hunt, T. (Nucl. Acids Res. 14 (16): 6433-6451 (1986); Sartorius, C. and Franklin, R.M. (Nucl. Acids Res. 19 (7): 1613-1618 (1991) and Krebs-2 cell-free system (Miroshnichenko, N.A. et al. (FEBS 234 (1): 65-68 (1988)).
  • Cellular systems include Xenopus oocytes (Dagle, J.M., et al. (Nucl. Acids Res. 18 (16): 4751- 4757 (1990)), and many cultured cell lines, for example HL-60 (Holt, J.T., et al. (Mol. Cell. Biol. 8 (2): 963-973 (1988)), and T15, N-ras transformed NIH3T3 cells (Tidd, D.M., et al. (Anti-Cancer Drug Design 3: 117-127 (1988)). There are also a limited number of in vivo examples (Wickstrom, E. et al. (FASEB J. 5: A1443 (1991)).
  • ODNs Antisense oligodeoxynucleotides
  • bp base pairs
  • an ODN must be specific for its target sequence, and it has been calculated statistically that a 14mer is the minimum length required to obtain only one exact match within the transcripts from the human genome (Ts'o, P.O.P., et al. (Biological Approaches to the Controlled Delivery of Drugs 507: Ann. N.Y. Acad. Sciences (1987)).
  • antisense technology has had the greatest impact in horticulture where antisense RNA produced in transfected cells has been used to modify petal colouration in Petunia by targeting Chalcone synthetase (Van der Krol, A.R., et al. (Nature 333: 866-869 (1988)), and to control tomato ripening by targeting the rate-limiting enzyme in the biosynthetic pathway of ethylene (Oeller, P. ., et al. (Science 254: 437- 439 (1991)).
  • oligonucleotides are not readily taken up by tissues and cells.
  • Approaches to improving cellular uptake include conjugation to poly(L- lysine) (Leonetti, J.P. et al. Gene 72: 323-332 (1988)), and inclusion of oligonucleotides in liposomes (Loke, S.L. et al.
  • antisense oligonucleotides are rapidly degraded by nuclease activity in serum and in cells.
  • the predominant nuclease activity affecting antisense efficacy is 3'-5' directed exonuclease activity.
  • the problem of degradation has been partially resolved by modifying the exposed phosphate backbone of oligonucleotides in order to alter nuclease recognition and therefore confer some measure of resistance to degradation.
  • Backbone modifications include methylphos- phonate ODNs, where an oxygen of the phosphate group is replaced by a -CH3 group (Miller, P.S. et al. (Biochemistry 20 (7): 1874-1880 (1981); Tidd, D.M. and arenius, H.M. (Br. J. Cancer 60: 343-350 (1989)), and phosphorothioate ODNs, where an oxygen is replaced by a sulphur (Stec, W.J. et al.
  • a further example is the addition of a 3' anthraquinone pseudonucleoside, which improves the strength of hybridisation without reducing specificity and also confers protection against exonuclease degradation (Lin, K.Y. and Matteucci, M. (Nucl. Acids Res. 19 (11): 3111-3114 (1991)).
  • Synthetic oligonucleotide analogues have the disadvantage of potential toxicity to cells.
  • the present invention is therefore directed to ways of stabilising oligonucleotides against nuclease degradation.
  • the present invention provides an - open oligodeoxynucleotide for inhibiting or modifying gene expression in vivo or in vitro comprising an antisense sequence of nucleotide bases and further nucleotides at one or both ends of the antisense sequence, one or both of the said further nucleotide sequences being capable of forming a secondary structure conferring at least partial nuclease resistance on the oligodeoxynucleotide.
  • An “open” oligodeoxynucleotide may be single stranded or double stranded. However there will always be free 3' and 5' ends.
  • a “closed” oligodeoxynucleotide may be single or double stranded but there will not be any free ends.
  • a “closed” oligodeoxynucleotide may be termed "ligated" in some references.
  • the term in vivo above is intended to cover living organisms, tissues and cells whereas in vitro is intended to cover cell extracts and cell free translation systems.
  • the inhibition or modification of gene expression will usually involve duplex formation between the oligodeoxynucleotide and the RNA or DNA sequence of interest to give an inhibition or modulation of translation.
  • Also possible is the inhibition or modulation of gene transcription by the formation of triplexes comprising an oligonucleotide and a double stranded duplex of DNA (Yoon et al. Proc Naltl. Acad Sci USA 89:3840- 3844(1992)).
  • the formation of triple helices has the potential to alter gene transcription ( Le Doan et al. Nucl. Acids Res.
  • triple helices can occur at regulatory regions of genes, these regions being the targets for sequence-specific DNA binding proteins.
  • the DNA binding proteins have the potential to activate or repress transcription from gene promoters.
  • Triple helix formation at sequence motifs recognised by DNA binding proteins may allow modulation of gene expression in order to effect upregulation by inhibiting the binding of a repressor or downregulation by inhibiting the binding of an activating protein.
  • Triple helices are formed by Hoogsteen- type hydrogen bonds between thymine and protonated cytosines with Watson-Crick A.T. and G.C. pairs respectively.
  • An oligodeoxynucleotide in accordance with the invention can therefore be used to inhibit the binding of a DNA binding protein to its binding site on DNA.
  • Oligodeoxynucleotides in accordance with the invention also include so-called chimeric oligodeoxynucleotides in which some or all of the nucleotide bases or part of the nucleic acid back-bone are chemically modified for stability against nuclease degradation or for other desirable properties such as good cellular uptake.
  • the modified bases may be present as a sequence or as isolated bases within a sequence.
  • An oligodeoxynucleotide may be provided in which all the bases are chemically modified.
  • the bases forming the secondary structure may also be modified. Chemically modified back-bones and bases include phosphorothioates, methylphosphonates and 3' anthraquinone pseudonucleosides.
  • Oligodeoxynucleotides in accordance with the invention may also be conjugated to poly( -L-Lysine) or incorporated in liposomes in order to improve their cellular uptake.
  • the invention includes an open oligodeoxynucleotide for use in the treatment of the human or animal body by therapy or diagnosis by inhibition of the expression of a gene product, the said oligodeoxynucleotide comprising an antisense sequence of nucleotide bases and further nucleotides at one or both ends of said antisense sequence one or both of the further nucleotide sequences being capable of forming a secondary structure conferring at least partial nuclease resistance on the oligodeoxynucleotide.
  • the said further nucleotide sequence forming the secondary structure preferably comprises at least 5 nucleotide residues.
  • the secondary structure comprises eight nudeotide residues.
  • the secondary structure may comprise up to 100 nucleotide residues.
  • the secondary structure is a hairpin loop.
  • the hairpin loop structure has a portion where nucleotide bases are paired together and a portion where they are unpaired.
  • the unpaired portion is generally kinked or looped back on itself.
  • the paired portion forms a double stranded stem which supports the loop.
  • the part of the sequence actually forming the loop may itself undergo non Watson-Crick type base pairing.
  • the said further nucleotide sequence forming the hairpin loop structure is preferably 5 ' GCGAAAGC 3 ' . This structure has been found to be particularly effective and is the most preferred structure.
  • the said further nucleotide sequence forming the hairpin loop structure may be 5 ' GCGTTCGC 3 ' .
  • the said further nucleotide sequence forming the hairpin loop structure may be 5 ' GCGTAAGC 3 ' .
  • the oligodeoxynucleotide preferably comprises more than 12 nucleotide residues.
  • the invention includes an open oligodeoxynucleotide comprising a sequence of more than twelve nucleotide residues and which has further nucleotide residues which form a secondary structure at one or both ends of the said sequence, the secondary structure conferring at least partial nuclease resistance on the oligodeoxynucleotide.
  • the oligodeoxynucleotide is capable of hybridising with at least a portion of a selected mRNA species.
  • the invention includes any oligodeoxynucleotide as herein-before defined in a pharmaceutically acceptable carrier.
  • the oligodeoxynucleotide may be incorporated in liposomes.
  • Such preparations include topical preparations e.g. ointments or creams where application of the oligodeoxynucleotide is to be made to the skin.
  • the oligodeoxynucleotides in accordance with the invention may include at least one chemically modified nucleotide base.
  • the oligodeoxynucleotides may have at least a portion of the sugar phosphate backbone modified chemically.
  • the invention also includes an oligodeoxynucleotide as herein defined for the manufacture of a medicament for use in a method of medical treatment of an organism, the said treatment involving blocking the expression of a selected gene or selected genes.
  • the stable oligodeoxynucleotides of the present invention are not restricted in their use to the treatment of human or animal diseases. They will of course be of widespread application wherever nuclease resistant oligodeoxynucleotides are required. For example the modification or inhibition of gene expression in plants and fungi, bacteria and other prokaryotes. Another area where the stable oligodeoxynucleotides of the invention will be of use is in diagnostics where crude cell extracts or extracelluar material could be screened using a labelled oligodeoxynucleotide probe which hybridises to a specific base sequence. The probe would carry at least one stabilising secondary structure.
  • Figure 1 is an autoradiograph of the Northern blot described in Example 1.
  • Figure 2 shows the sequences and target sites of anti- sense oligodeoxynucleotides (ODNs ) around an AUG start codon associated with the sequence for vaccinia virus DNA polymerase.
  • ODNs anti- sense oligodeoxynucleotides
  • Figure 3 is the thermal denaturation curve used for determining T m values in Example 1.
  • Figure 4 is an autoradiograph of the 15% polyacrylamide (PAGE) gel described in Example 1.
  • Figure 5 is a histogram representing the results of the experiments described in Example 2.
  • Figure 6 is a histogram representing the results of further experiments described in Example 2.
  • Figure 7 shows an autoradiograph of the 20% PAGE (7M urea) gel described in Example 3.
  • Figure 8 shows sequences of ODNs tagged with the 3' hairpin sequences described in Example 4.
  • Figure 9 shows autoradiographs of the 20% PAGE gels (7M urea) described in Example 4.
  • Figure 10 is an autoradiograph of the 20% PAGE (7M urea) gel described in Example 4.
  • Figure 11 shows an autoradiograph of the PAGE gel described in Example 5.
  • Figure 12 shows an autoradiograph of the PAGE gel described in Example 6.
  • Figure 13 is the graph used for determining T values described in Example 7.
  • Figure 14 is a histogram representing the results of the experiment described in Example 8.
  • Figure 15 comprises histograms representing the results of the experiments described in Example 8.
  • Vaccinia virus is a member of the poxviridiae family and is generally regarded as the prototype poxvirus.
  • the virions are large (200-400 n ) and the 186 kb double stranded DNA genome is contained in a central core.
  • the genome is characteristic of the poxviruses in that it is A/T rich, has inverted terminal repeats, covalently cross-linked ends and termini which exist as hairpin loops.
  • the virus is adsorbed on to cells before entering. Once inside a cell the virus particle uncoats and the early genes are transcribed. The viral DNA is then replicated prior to transcription of late genes. Mature virus particles, once assembled, migrate to the Golgi apparatus where they become enveloped in a double membrane before release from cells.
  • Vaccinia virus is easily grown in cell culture and so antisense ODNs can be tested against live virus in a cell culture system, allowing quantitation of the effect of antisense ODN on viral titre.
  • the entire sequence of the Copenhagen strain of vaccinia is known (Goebel, S.J. et al. (Virology 179: 247-266 (1990)), and many sequences have been obtained for individual genes of other strains, such as WR.
  • Vaccinia encodes several virus specific proteins which provide suitable antisense targets (Moss, B. ("Replication of Poxviruses in Virology"; 685-703 Fields, B.N. et al eds. Raven Press, New York (1985)).
  • DNA polymerase examples are the DNA polymerase, RNA polymerase, and topioisomerases.
  • DNA polymerase gene which was sequenced in the WR strain several years ago (Earl, P.L. et al. (Proc. Natl. Acad. Sci. USA 83: 3659-3663 (1986)), is the most appropriate target due to its absolute requirement for virus proliferation (Moss, B. and Cooper, N. (J. Virol. 43: 673-678 (1982)), and the fact that unlike the RNA polymerase the protein is not present in the virus particle at the start of the life cycle.
  • the 1.1 kb portion of the 5' end of the vaccinia virus WR strain was cloned and transcribed in vitro.
  • the 5 ' region of the WR vaccinia virus DNA polymerase gene was amplified by Polymerase Chain Reaction (PCR). An ATG triplet upstream of a target initiation codon is removed and a stop codon introduced in the resultant 1107 base pair fragment in order to enhance translation in a rabbit reticulocyte lysate.
  • the PCR product was cloned into the transcription vector Bluescript ( Stratagene) to produce the construct pBS-VACC(2).
  • a 1.05 kb RNA corresponding to the cloned portion of the DNA polymerase coding region was transcribed in vitro as described below.
  • PBS-VACC(2) was linearised by overnight digestion with BamHl to produce a template for the production of run off transcripts. Transcriptions were carried out using a mCAP mRNA capping kit (Stratagene) , this includes a 5'7meGpppG5' cap analogue which is used to initiate transcription, the resultant RNA being more efficiently translated in an in vitro system. T3 RNA polymerase was used for transcription of the sense RNA. The resultant RNA was 1.05 kb which was the expected length and this was detectable on a Northern blot using J ⁇ * 7P ⁇ ATP end-labelled ODN (JC3 sequence shown in Figure 2 ) as a probe ( see Figure 1 ) .
  • RNA of the correct size is transcribed in vitro from the construct pBS-VACC(2). This is detected on a Northern olot using the antisense oligonucleo ⁇ tide JC3 as a probe, illustrating that the RNA does contain the relevant target sequence and that the oligonucleotide will bind to this under stringent conditions.
  • the various antisense ODNs and control ODNs used in these Examples were prepared by a standard method of oligonucleotide synthesis, familiar to the average worker skilled in the art. ODNs were synthesised on an ABI 380B (Applied Biosystems) using standard techniques. ODNs were purified on NAP10 columns (Pharmacia) or by reverse phase chromatography on an OPC Column (Applied Biosystems). The purity of the ODNs was analysed by capillary electrophoresis.
  • ODN sequences and target sites are shown in Figure 2. Characters which are underlined indicate the position where base changes have been made in the cloned sequence from those in the wild type sequences (shown), to those shown in the PCR primers VACC(l) and VACC(2). All the antisense ODNs are complementary to the initiation codon region (AUG) of the DNA polymerase gene. JC2 is a 14mer which spans a base change engineered into the cloned sequence to enhance translation. Therefore, JC2 (mod) was synthesised for use in the in vitro translation assay. JC3 is an 18mer ODN which avoids this mismatch. JC4 is a second 18mer complementary to a region of RNA lying nine bases upstream of the JC3 target site, encompassing the JC2 target site. Binding kinetics for the hybridisation of the antisense
  • ODNs to the in vitro transcript were determined by quantitation of ° ⁇ v ⁇ P end-labelled ODN released from the target
  • T m values that is the temperature at which 50% of the oligonucleotide has dissociated from the RNA, were determined as follows.
  • RNA denaturing buffer (20% (v/v) formaldehyde, 6xSSC) and denatured by 10 min incubation at 65 ⁇ C, freezing at -20 ⁇ C overnight, followed by a second 65 ⁇ C incubation.
  • lOOng of denatured RNA was fixed to 0.5 cm diameter discs of Hybond-N (Amersham) by 15 seconds UV illumination and 2 hours baking at 80°C.
  • Discs were prehybridised for 2 hrs at 65°C in 2 ml of ODN prehybridisation solution (6xSSC, 0.01 M Sodium phosphate pH 6.8, ImM EDTA pH8.0, 0.5% (w/v) SDS, lOOmg/ml denatured frag ented salmon sperm DNA, 0.1% (w/v) Nonfat dried milk).
  • ODN prehybridisation solution 6xSSC, 0.01 M Sodium phosphate pH 6.8, ImM EDTA pH8.0, 0.5% (w/v) SDS, lOOmg/ml denatured frag ented salmon sperm DNA, 0.1% (w/v) Nonfat dried milk.
  • the labelled ODN was then added to the prehybridisation solution to give a concentration of 5 pmol/ml.
  • Hybridisation was 2 hours at 65°C, cooling slowly overnight to room temperature for annealing of ODNs to the RNA.
  • PB near physiological buffer
  • the disintegrations per minute for each wash were determined by Cerenkov counting for 60 sec in a LKB counter. The percentage of the total counts released at each temperature was then plotted and used to determine the temperature at which 50% of the labelled ODN has dissociated from the bound RNA; that is the T m value.
  • Figure 3 shows that the T m values for the hybridisation of the ODNs to the target RNA in 3xSSC are 41°C for JC2, 57°C for JC3 and 53°C for JC4.
  • RNA produced in vitro was translated into its encoded proteins in a rabbit reticulocyte lysate.
  • SDS PAGE analysis of translation products showed a major protein band of about 40KD.
  • the ability of the antisense ODNs to inhibit in vitro translation was tested in relation to control ODNs by incubation of ODNs with RNA prior to addition of the lysate. 50 ng of the in vitro transcript was used in each in vitro translation reaction. Translation reactions were carried out using rabbit reticulocyte lysate (GIBCO-BRL). 10 ⁇ l reaction volumes comprised by volume 33% lysate, 8% IM potassium acetate (pH 7.2), 10% translation mixture (- methionine, GIBCO-BRL) in the presence of 35 S methionine (Amersham).
  • RNA was made up to 5 ⁇ l with diethylpyrocarbonate (DEPC) treated H 2 0, before addition of the components listed above.
  • DEPC diethylpyrocarbonate
  • the RNA and ODN were mixed at room temperature prior to addition of the translation reagents. Reactions were incubated at 30°C for 1 hour. Translation products were quantitated by TCA precipitation of synthesised proteins and scintillation counting. The products were analysed by electrophoresis on denaturing 15% SDS-PAGE gels.
  • Figure 4 shows analysis of in vitro translation products from various reactions on a 15% SDS PAGE gel.
  • CV-1 cells were infected with vaccinia virus and then oligonucleotides were added.
  • the vaccinia virus was harvested from these cells after 48 hrs and titred in fresh CV-1 cells to determine the total yield of virus.
  • the results for CV-ls infected at a multiplicity of lpfu/cell are shown in Figure 6.
  • FCS minimal essential medium
  • ODNs in MEM showed no degradation. In 2.5% FCS there was significant degradation of the ODN indicated by the "ladder" of smaller molecular weight components and the depletion of the starting molecular weight band. At L0% FCS there was significant degradation after only 2 hr, equivalent to that seen after 20 hr in 2.5% FCS.
  • the ODNs shown in Figure 8 were synthesised using a standard method of oligonucleotide synthesis.
  • the bases which are underlined form hairpin loop type secondary structures.
  • Two different structures were used being designated HPl and HP2. These were synthesised as part of the antisense sequence JC4 shown in Figure 2.
  • the sequence JC4SHP2 is a control wherein the sequence of the antisense ODN is scrambled.
  • JC4HP2S is also a control wherein the HP2 sequence is scrambled and so should form no secondary structure.
  • the AS3HP2 sequence is an antisense ODN to the E6 gene of Human Papilloma Virus HPV16 including the HP2 hairpin loop.
  • HP3 differs from HP2 in that there are two adenosine residues and one thymidine residue, rather than three adenosine residues.
  • Figure 10 shows a 20% polyacylamide 7M urea gel analysis of ODN degradation products produced from 5' end-labelled antisense ODNs targetted to HPV16 E6, (HPVAS3 and HPVAS3HP2) in MEM + 10% foetal calf serum at 37°C.
  • the gel shows that HPVAS3HP2 is stable in 10% FCS for up to 19 hours. There are no apparent products of 3 ' exonuclease activity meaning that the HP2 structure is unexpectedly more stable than HPl.
  • HP2 shows a many-fold greater stability than the unmodified ODNs.
  • Phosphodiesterase I releases 5' mononucleo- tides from the 3' end of oligonucleotides (Hayashi, 0. TIBS . 1:9(1976)). The enzyme is therefore a 3'-5' exonuclease.
  • the Phosphodiesterase I reactions were conducted as follows.
  • the following components were added to make a final reaction volume of lO ⁇ l; 10X Reaction Buffer (0.33M Tris-acetate, 0.66M Potassium acetate, 0.1M Magnesium acetate), 0.5ng 32 P end- labelled DNA oligodeoxynucleotide, 100 ng unlabelled DNA oligodeoxynucleotide as a competitor, and 0.5ng-5ng of Phosphodiesterase I.
  • the components were mixed and samples incubated in a water bath at 37 ⁇ C. The reactions were visualised by electrophoresis on a 7M Urea 20% polyacrylamide gel followed by autoradio-graphy of the wet gel.
  • Lanes 9-11 0.5ng and lanes 12-14 5ng of added enzyme for the above stated time intervals clearly show HPVAS3 as a degraded ladder of smaller oligonucleotides. Lanes 12-14 show that HPVAS3HP2 is clearly resistant to 3'-5' attack by Phosphodiesterase I.
  • the experiment is there- fore an assay for the presence of a 3' hairpin loop on an oligodeoxynucleotide.
  • the oligodeoxynucleotides compared were JC4HP2 and JC4HP2S.
  • 20 ⁇ l final reaction volumes contained 10X Klenow buffer, 2 ⁇ l 0.5mM dNTP's, 0.5ng kinase labelled DNA oligonucleotide and 1 unit of Klenow enzyme.
  • the void reaction volume was made up with deionised, distilled water. The reactions were incubated for various times between 5 and 15 minutes, and stopped by the addition of stop-mix and loaded on to a (7M Urea) 20% polyacylamide gel.
  • Lane 1 is the control reaction; JC4HP2 + no Klenow enzyme.
  • Lanes 2-4 increasing time of incubation with Klenow enzyme with JC4HP2, 5, 10, 15 minutes respectively.
  • Lane 5 control reaction, no Klenow enzyme added to JC4HP2S.
  • Lane 6 incubation of JC4HP2S with Klenow enzyme for 15 minutes.
  • a higher molecular weight band is observed in tracks 2-4 where JC4HP2 is incubated with Klenow enzyme. Presumably this band is the result of Klenow enzyme forming a double-stranded oligodeoxynucleotide from the single-stranded hairpin loop intermediate form.
  • the scrambled hairpin sequence oligodeoxynucleotide JC4HP2S in lane 6 does not show any double-stranded forms.
  • JC4HP2S Upon exposure to Phosphodiesterase I JC4HP2 is found to be resistant whereas JC4HP2S is degraded.
  • the nuclease resistance of sequence modified oligodeoxy ⁇ nucleotides would appear to be due primarily to the presence of the hairpin loop structure.
  • the sequence of the hairpin loop used in these examples was carefully chosen in order to fulfil two major require ⁇ ments; it must form a stable hairpin at 37°C, and it must be compact enough, not to interfere with hybridisation between the oligonucleotide and its target sequence. It also should not compromise the entry of oligonucleotides into the cell.
  • the sequence of 5 ' GCGAAAGC3 ' has unusual characteristics in that it forms an extra-ordinarily stable secondary structure in the form of a hairpin loop. Significantly, its melting temperature (the temperature where half of the oligonucleotide is found in the random coil conformation and half in the hairpin conformation) is 76.5°C under normal conditions and in 7M Urea, 53°C.
  • oligonucleotide containing the sequence dGCGAAAGC would be adopting the structure of a hairpin loop. Consequently, sequence modified oligonucleotides were chemically synthesised in the 3'-5' direction, incorporating the hairpin loop at the 3' end of a defined single-stranded region.
  • sequence modified oligonucleotides were chemically synthesised in the 3'-5' direction, incorporating the hairpin loop at the 3' end of a defined single-stranded region.
  • These oligo ⁇ nucleotides, prior to undergoing incubation in serum or with enzymes are heated to 95°C for 15 minutes and allowed to cool to room temperature ( Xodo et al. Biochemistry 27:6321-6326 (1988)). This procedure firstly forces all oligonucleotides into the random coil conformation.
  • the oligo ⁇ nucleotides Upon cooling, the oligo ⁇ nucleotides undergo a transition which is directed by their melting temperature to the hairpin state.
  • the hairpin loop containing oligonucleotides can be stored for long periods at -20°C. The heating and cooling of the hairpin oligonucleotides before use helps to ensure that all the hairpin sequences are in the required conformation and hence helps to stabilise their resistance to nuclease attack.
  • hairpin loops does not sterically hinder hybridisation of the antisense ODN to its target RNAs. This is shown firstly by the T m values for these hybridisations, and secondly by the fact that the ODNs can prevent translation of the target RNA in a rabbit reticulocyte lysate.
  • T m determinations were conducted in a near physiological buffer. T m determinations for JC4, JC4HP1 and JC4HP2 are shown in Figure 13.
  • the graph shows the dissociation of 32 P end-labelled ODNs: JC4, JC4HP1 and JC4HP2 from target RNA with temperature in a near physiological buffer.
  • the released radiolabelled ODN was determined by Cerenkov counting at 2.5°C temperature intervals.
  • JC4HP2 shows a release profile almost identical to that of the normal ODN, JC4, indicating that the modification has had little effect on hybridisation to the target RNA.
  • JC4HP1 however leads to a 6°C increase in the determined T m , but this may be due to "breathing" of this hairpin structure allowing some nonspecific binding of the ODN 3' extension to the RNA.
  • JC4HP1 prevents translation of RNA coding for the 5 ' end of the vaccinia virus DNA polymerase in rabbit reticulocyte lysate, see Figure 4, lanes 8 and 9. AT 25 ⁇ M JC4HP1 does not completely prevent translation as seen with JC4, but inhibition is total at 50 ⁇ M. Similar results are seen with JC4HP2 - data not shown.
  • the hairpin ODNs were tested in the cell culture system described in Example 2.
  • the effective concentration of ODN used was 25 ⁇ M.
  • CV-ls were infected at a specific viral infectivity of 0.0005 pfu/cell and the virus yield after 48 hours determined.
  • the error bars indicate one standard deviation of the triplicate titres.
  • yields of vaccinia virus were reduced to 61%, 81% and 45% of the control value for JC3, JC4 and JC4HP1 respectively.
  • the addition of the HPl structure appears to enhance the action of JC4, presumably by increasing the half life of the ODN.
  • Tests of JC4HP2 on vaccinia virus in cell culture were also carried out using a specific viral infectivity of 0.0005 pfu/cell and an effective concentration of 25 ⁇ M ODN.
  • Figure 15 the yield of vaccinia virus from CV-1 cells is shown for 24 hr and 48 hr post-infection, (a) is extracelluar virus harvested from the cell culture medium, and (b) is intra ⁇ cellular virus harvested from the cells. Error bars indicate the standard deviation of triplicate titres.
  • a reduced virus yield was seen in intracellular virus at 24 hours and in both extracelluar and intracellular virus at 48 hours, in the presence of 25 ⁇ M JC4HP2 when compared to the ODN free controls. These reductions are 5, 2.7 and 3.2 fold respectively (i.e. 20%, 37% and 31% of the controls). From these preliminary results the HP2 modification therefore appears to enhance the antisense effect of JC4 in this system.
  • HUMAN PAPILLOMA VIRUS HPV16 Oligonucleotides having nucleic acid base sequences antisense to parts of the E6 or E7 genes of human papilloma virus (HPV16) are provided by a standard method of oligonucleotide synthesis.
  • each oligonucleotide has at its 3' end a hairpin loop forming sequence.
  • the hairpin loop forming sequence is one of 5*GCGTTCGC3' , 5'GCGAAAGC3' or 5'GCGTAAGC3' .
  • the hairpin loop forming sequence renders the oligo ⁇ nucleotides antisense to E6 or E7 more resistent to nuclease degradation.
  • the respective antisense oligonucleotides are able to reduce the expression of E6 and E7 genes in a cell free translation system.
  • the stabilised antisense oligo ⁇ nucleotides should be capable of reducing the expression of HPV16 genes integrated into the genomes of SiHa or CaSki human cell culture lines.

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Abstract

Procédé permettant de rendre des oligodésoxynucléotides plus résistants à la décomposition par des nucléases, selon lequel on incorpore une séquence de base au niveau de l'extrémité 3' qui forme une structure secondaire du type boucle en épingle à cheveux. La séquence de boucle en épingle à cheveux forme une structure extrêmement stable qui est constituée d'une tige à deux brins et d'une région de boucle, et qui se situe à l'extrémité 3' par rapport à la séquence antisens à un seul brin. Les structures de boucle en épingle à cheveux sont avantageusement utilisées pour stabiliser des oligodésoxynucléotides antisens qui sont utilisés pour bloquer l'expression de gènes $(in vivo) et $(in vitro).
PCT/GB1993/002409 1992-11-24 1993-11-23 Oligonucleotides stables WO1994012633A1 (fr)

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WO1994023026A1 (fr) * 1993-03-26 1994-10-13 Genset Oligonuclotides agrafes et semi-agrafes, procede de preparation et applications
US5545729A (en) * 1994-12-22 1996-08-13 Hybridon, Inc. Stabilized ribozyme analogs
US5627055A (en) * 1994-09-29 1997-05-06 Hybridon, Inc. Finderons and methods of their preparation and use
US5650502A (en) * 1994-11-09 1997-07-22 Hybridon, Inc. Ribozyme analogs having rigid non-nucleotidic linkers
DE19631919A1 (de) * 1996-08-07 1998-02-12 Deutsches Krebsforsch Anti-Sinn-RNA mit Sekundärstruktur
WO1999002673A2 (fr) * 1997-07-10 1999-01-21 Genesense Technologies, Inc. Sequences oligonucleotidiques antisens servant d'inhibiteurs de micro-organismes
WO2000053745A1 (fr) * 1999-03-09 2000-09-14 Bioalliance Pharma (S.A.) Oligonucleotides contenant une sequence antisens stabilises par une structure secondaire et compositions pharmaceutiques les contenant
WO2001027146A2 (fr) 1999-10-12 2001-04-19 Chemocentryx, Inc. Recepteur de chemokine
US6451563B1 (en) * 1998-06-15 2002-09-17 Mologen Forschungs-, Entwicklungs- Und Vertriebs Gmbh Method for making linear, covalently closed DNA constructs
EP1333094A2 (fr) 1996-10-01 2003-08-06 Geron Corporation Sous-unité catalytique de la télomérase humaine
WO2005030960A1 (fr) * 2003-09-30 2005-04-07 Anges Mg, Inc. Oligonucleotide du type en agrafe et medicament comprenant celui-ci
EP1584681A2 (fr) * 1997-07-10 2005-10-12 GeneSense Technologies Inc. Séquences oligonucléotidiques antisens servant d'inhibiteurs de micro-organismes
WO2006086108A2 (fr) 2005-01-10 2006-08-17 Regents Of The University Of California Utilisation d'inhibiteurs de l'epoxyde hydrolase soluble pour synergiser l'activite d'inhibiteurs de cox et de 5-lox
EP1838875A2 (fr) * 2004-12-30 2007-10-03 Todd M. Hauser Compositions et procedes pour la modulation de l'expression genique par oligonucleotides a autoprotection
WO2011005779A2 (fr) 2009-07-07 2011-01-13 University Of Southern California Biomarqueurs pour la détection précoce de maladies auto-immunes
EP2311949A2 (fr) 2003-12-03 2011-04-20 Coda Therapeutics (NZ) Ltd Composés anti-sens ciblés contre les connexines et procédés d'utilisation associés
US8247384B2 (en) 2006-11-15 2012-08-21 Coda Therapeutics, Inc. Methods and compositions for wound healing
EP2510939A1 (fr) 2005-02-03 2012-10-17 Coda Therapeutics Limited Composés peptidomimétiques à activité anti-connexine et utilisations thérapeutiques
WO2013138118A1 (fr) 2012-03-14 2013-09-19 The Regents Of The University Of California Traitement de troubles inflammatoires chez des mammifères non humains
WO2014100602A1 (fr) 2012-12-20 2014-06-26 Hospital For Special Surgery Traitement de pathologies dépendant du récepteur de l'egf
WO2015048531A1 (fr) 2013-09-26 2015-04-02 Beth Israel Deaconess Medical Center, Inc. Inhibition de la sgk1 dans le traitement d'affections cardiaques
US10465188B2 (en) 2014-08-22 2019-11-05 Auckland Uniservices Limited Channel modulators
US11466069B2 (en) 2017-04-28 2022-10-11 Auckland Uniservices Limited Methods of treatment and novel constructs

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Cited By (44)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994023026A1 (fr) * 1993-03-26 1994-10-13 Genset Oligonuclotides agrafes et semi-agrafes, procede de preparation et applications
US5627055A (en) * 1994-09-29 1997-05-06 Hybridon, Inc. Finderons and methods of their preparation and use
US5646021A (en) * 1994-09-29 1997-07-08 Hybridon, Inc. Finderons and methods of their preparation and use
US5679554A (en) * 1994-09-29 1997-10-21 Hybridon, Inc. Finderons and methods of their preparation and use
US5700923A (en) * 1994-09-29 1997-12-23 Hybridon, Inc. Finderons and methods of their preparation and use
US5650502A (en) * 1994-11-09 1997-07-22 Hybridon, Inc. Ribozyme analogs having rigid non-nucleotidic linkers
US5679555A (en) * 1994-11-09 1997-10-21 Hybridon, Inc. Method of using ribozyme analogs having rigid, non-nucleotidic molecular linkers
US5545729A (en) * 1994-12-22 1996-08-13 Hybridon, Inc. Stabilized ribozyme analogs
DE19631919A1 (de) * 1996-08-07 1998-02-12 Deutsches Krebsforsch Anti-Sinn-RNA mit Sekundärstruktur
DE19631919C2 (de) * 1996-08-07 1998-07-16 Deutsches Krebsforsch Anti-Sinn-RNA mit Sekundärstruktur
EP1333094A2 (fr) 1996-10-01 2003-08-06 Geron Corporation Sous-unité catalytique de la télomérase humaine
EP2213740A1 (fr) 1996-10-01 2010-08-04 The Regents of the University of Colorado Sous-unité catalytique de la télomérase humaine
EP1584681A2 (fr) * 1997-07-10 2005-10-12 GeneSense Technologies Inc. Séquences oligonucléotidiques antisens servant d'inhibiteurs de micro-organismes
EP1584681A3 (fr) * 1997-07-10 2005-11-09 GeneSense Technologies Inc. Sequences oligonucleotidiques antisens servant d'inhibiteurs de micro-organismes
WO1999002673A3 (fr) * 1997-07-10 1999-04-01 Genesense Technologies Inc Sequences oligonucleotidiques antisens servant d'inhibiteurs de micro-organismes
WO1999002673A2 (fr) * 1997-07-10 1999-01-21 Genesense Technologies, Inc. Sequences oligonucleotidiques antisens servant d'inhibiteurs de micro-organismes
US6451563B1 (en) * 1998-06-15 2002-09-17 Mologen Forschungs-, Entwicklungs- Und Vertriebs Gmbh Method for making linear, covalently closed DNA constructs
FR2790757A1 (fr) * 1999-03-09 2000-09-15 Bioalliance Pharma Oligonucleotides contenant une sequence antisens stabilises par une structure secondaire et compositions pharmaceutiques les contenant.
WO2000053745A1 (fr) * 1999-03-09 2000-09-14 Bioalliance Pharma (S.A.) Oligonucleotides contenant une sequence antisens stabilises par une structure secondaire et compositions pharmaceutiques les contenant
US7022832B2 (en) 1999-03-09 2006-04-04 Bioalliance Pharma (S.A.) Oligonucleotides containing an antisense sequence stabilized by a secondary structure, pharmaceutical compositions containing them and method of blocking gene expression using them
WO2001027146A2 (fr) 1999-10-12 2001-04-19 Chemocentryx, Inc. Recepteur de chemokine
WO2005030960A1 (fr) * 2003-09-30 2005-04-07 Anges Mg, Inc. Oligonucleotide du type en agrafe et medicament comprenant celui-ci
US7595301B2 (en) 2003-09-30 2009-09-29 Anges Mg, Inc. Staple type oligonucleotide and drug comprising the same
EP2311949A2 (fr) 2003-12-03 2011-04-20 Coda Therapeutics (NZ) Ltd Composés anti-sens ciblés contre les connexines et procédés d'utilisation associés
EP2314689A2 (fr) 2003-12-03 2011-04-27 Coda Therapeutics (NZ) Ltd Composés anti-sens ciblés contre les connexines et procédés d'utilisation associés
EP1838875A2 (fr) * 2004-12-30 2007-10-03 Todd M. Hauser Compositions et procedes pour la modulation de l'expression genique par oligonucleotides a autoprotection
EP1838875A4 (fr) * 2004-12-30 2010-08-25 Todd M Hauser Compositions et procedes pour la modulation de l'expression genique par oligonucleotides a autoprotection
WO2006086108A2 (fr) 2005-01-10 2006-08-17 Regents Of The University Of California Utilisation d'inhibiteurs de l'epoxyde hydrolase soluble pour synergiser l'activite d'inhibiteurs de cox et de 5-lox
EP2662088A2 (fr) 2005-02-03 2013-11-13 Coda Therapeutics Limited Composés anti-connexine et utilisations associées
US9248141B2 (en) 2005-02-03 2016-02-02 Coda Therapeutics, Inc. Methods of treatment by administering anti-connexin proteins and mimetics
EP2510939A1 (fr) 2005-02-03 2012-10-17 Coda Therapeutics Limited Composés peptidomimétiques à activité anti-connexine et utilisations thérapeutiques
EP2510938A1 (fr) 2005-02-03 2012-10-17 Coda Therapeutics Limited Composés anti-connexine et utilisation pour le traitement de désorders vasculaires
EP2510937A1 (fr) 2005-02-03 2012-10-17 Coda Therapeutics Limited Composés anti-connexine et utilisations associées à une procédure de transplantation ou de greffe
US10201590B2 (en) 2005-02-03 2019-02-12 Ocunexus Therapeutics, Inc. Treatment of ocular disorders with anti-connexin proteins and mimetics
US9457044B2 (en) 2006-11-15 2016-10-04 Coda Therapeutics, Inc. Methods and compositions for wound healing
US8247384B2 (en) 2006-11-15 2012-08-21 Coda Therapeutics, Inc. Methods and compositions for wound healing
US10406174B2 (en) 2006-11-15 2019-09-10 Ocunexus Therapeutics, Inc. Methods and compositions for wound healing
WO2011005779A2 (fr) 2009-07-07 2011-01-13 University Of Southern California Biomarqueurs pour la détection précoce de maladies auto-immunes
WO2013138118A1 (fr) 2012-03-14 2013-09-19 The Regents Of The University Of California Traitement de troubles inflammatoires chez des mammifères non humains
WO2014100602A1 (fr) 2012-12-20 2014-06-26 Hospital For Special Surgery Traitement de pathologies dépendant du récepteur de l'egf
WO2015048531A1 (fr) 2013-09-26 2015-04-02 Beth Israel Deaconess Medical Center, Inc. Inhibition de la sgk1 dans le traitement d'affections cardiaques
US10465188B2 (en) 2014-08-22 2019-11-05 Auckland Uniservices Limited Channel modulators
US11401516B2 (en) 2014-08-22 2022-08-02 Auckland Uniservices Limited Channel modulators
US11466069B2 (en) 2017-04-28 2022-10-11 Auckland Uniservices Limited Methods of treatment and novel constructs

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