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WO1995028942A1 - Oligomeres non codants utilises pour inhiber les virus du papillome humains - Google Patents

Oligomeres non codants utilises pour inhiber les virus du papillome humains Download PDF

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Publication number
WO1995028942A1
WO1995028942A1 PCT/US1995/005179 US9505179W WO9528942A1 WO 1995028942 A1 WO1995028942 A1 WO 1995028942A1 US 9505179 W US9505179 W US 9505179W WO 9528942 A1 WO9528942 A1 WO 9528942A1
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WIPO (PCT)
Prior art keywords
oligomer
oligomer according
linkages
hpv
mrna
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PCT/US1995/005179
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English (en)
Inventor
Cristina Giachetti
James E. Marich
John A. Jaeger
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Genta Incorporated
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Publication date
Priority claimed from PCT/US1994/013387 external-priority patent/WO1995013834A1/fr
Application filed by Genta Incorporated filed Critical Genta Incorporated
Priority to AU25843/95A priority Critical patent/AU2584395A/en
Publication of WO1995028942A1 publication Critical patent/WO1995028942A1/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
    • 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
    • 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
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/15—Nucleic acids forming more than 2 strands, e.g. TFOs
    • 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
    • C12N2310/312—Phosphonates
    • C12N2310/3125—Methylphosphonates
    • 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
    • C12N2310/31—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
    • C12N2310/30—Chemical structure
    • C12N2310/31—Chemical structure of the backbone
    • C12N2310/316—Phosphonothioates
    • 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
    • C12N2310/321—2'-O-R Modification
    • 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
    • C12N2310/322—2'-R Modification

Definitions

  • Papillomaviruses are a group of small DNA viruses that induce warts (or papillomas) in a number of higher vertebrates, including humans. Although the viral nature of human warts has been known for many years, it has only recently been recognized that specific human papillomavirus ("HPV's”) are closely linked with certain human cancers most notably human cervical carcinoma. This finding has focused interest on the specific subgroup of HPV's associated with genital infections.
  • HPV's human papillomavirus
  • the papillomaviruses are reported to be highly species specific and to induce squamous epithelial tumors and fibroepithelial tumors in their natural hosts.
  • Bovine papillomavirus-1 (BPV-1) has been more fully characterized and has served as a prototype for studies on the transformation and molecular biology of human papillomaviruses. Significant differences exist between BPV-1 and the HPV's.
  • Certain proteins have been proposed as targets for antisense therapy of HPV-caused conditions. Certain of these targets have been proposed based on studies performed using BPV-1 and equivalent functions and properties have not necessarily been confirmed in all HPV's. In addition, other parameters to be considered in the selection of targets for antisense effect, such as mRNA sequence homology of target proteins between BPV and a HPV or the mRNA' s secondary structure (which strongly affect the accessibility of the target
  • Genital HPV's not associated with a risk for malignant progression include HPV-6b and HPV-11.
  • E7 protein In human papillomaviruses the E7 protein has been reported to have transcriptional modulating properties and to complex pl05-RB, a product of the retinoblastoma tumor suppressor gene. In HPV-16 and HPV-18, E7 is reported to encode transforming proteins, proteins which are multifunctional and possess both transcriptional modulatory and transformation properties similar to that adenovirus EIA. It has been reported that the E7 proteins of all the genital associated HPV s can complex pl05-RB in vitro regardless of their associated risk for malignant progression.
  • the E6 gene and its protein is also reported as associated with transformation in high risk human papilloma viruses, through interactions with p53.
  • HPV-6 and HPV-11 have been categorized as "low risk", in that patients
  • HPV's infected with these HPV's appear to be at significantly lower risk for malignant progression, it should be noted that such infections are not without risk for malignant progression and occasional tumors do contain these viral genomes that are transcriptionally active. Therefore, all genital HPV s should be considered capable of causing serious conditions. Accordingly, detection and control of HPV infections, especially those caused by genital HPV's is important.
  • the present invention is directed to antisense oligomers which are complementary to and which are capable of hybridizing with a target sequence of a mRNA or pre-mRNA of an human papillomavirus.
  • antisense oligomers which are complementary to and which hybridize with a portion of a mRNA or pre-mRNA encoding E1, E2, E6 or E7 gene of an HPV. These antisense oligomers interfere with and/or prevent expression of their target mRNA and thus, may be used for treatment and/or diagnosis of HPV infections as well as for research purposes.
  • oligomers complementary to a target sequence which is a portion of a HPV mRNA or pre-mRNA which encodes E1, E2, E6 or E7.
  • the target sequence is in the region of the initiation codon, more preferably from about -25 to about +35 relative to the initiation codon where the initiation codon is +1 to +3.
  • purine or “purine base” includes not only the naturally occurring adenine and guanine bases, but also modifications of those bases such as bases
  • pyrimidine or "pyrimidine base” includes not only the naturally occurring cytosine, uracil and thymine but also modifications to these bases such as 5-propynyluracil, 5-heteroaryluracils and analogs of pyrimidines such as reported heteroaromatic moieties.
  • nucleoside includes a nucleosidyl unit and is used interchangeably therewith, and refers to a subunit of a nucleic acid which comprises a 5-carbon sugar and a nitrogen-containing base.
  • the term includes not only those nucleosidyl units having A, G, C, T and U as their bases, but also analogs and modified forms of the naturally-occurring bases, including the pyrimidine analogs such as pseudoisocytosine and pseudouracil and other modified bases (such as 8-substituted purines).
  • the 5-carbon sugar is ribose; in DNA, it is a 2'-deoxyribose.
  • nucleoside also includes other analogs of such subunits, including those which have modified sugars such as 2'-O-alkyl ribose.
  • R is hydrogen or an alkyl or aryl group, and thus includes various example of phosphonate and phosphonothioate internucleosidyl linkages.
  • Suitable alkyl or aryl groups include those which do not sterically hinder the phosphonate linkage or interact with each other.
  • the phosphonate group may exist in either an "R" or an "S" configuration.
  • Phosphonate groups may be used as internucleosidyl linkages (or links) to connect nucleosidyl unit or a nucleosidyl unit and a non-nucleosidy monomeric unit.
  • the term “lower alkylphosphonate” refers to groups where X is oxygen and R is lower alkyl of 1 to 3 carbon atoms.
  • Methodylphosphonate refers to groups where X is oxygen and R is methyl.
  • phosphonothioate refers to those groups where X is sulfur.
  • lower alkylphosphonate refers to groups where X is oxygen and R is lower alkyl of 1 to 3 carbon atoms.
  • Method “Methylphosphonate” refers to groups where X is oxygen and R is methyl.
  • phosphonothioate refers to those groups where X is sulfur.
  • lower alkylphosphonate refers to groups where X is oxygen and R is lower alkyl of 1
  • alkylphosphonothioate refers to groups where X is sulfur and R is lower alkyl of 1 to 3 carbon atoms.
  • methylphosphonothioate refers to a
  • phosphodiester or “diester” refers to
  • non-nucleoside monomeric unit refers to a monomeric unit wherein the base, the sugar and/or the phosphorus backbone has been replaced by other chemical moieties.
  • nucleoside/non-nucleoside polymer refers to a polymer comprised of nucleoside and non-nucleoside monomeric units.
  • oligonucleoside or “Oligomer” refers to a chain of nucleosides which are linked by internucleoside linkages which is generally from about 4 to about 100 nucleosides in length, but which may be greater than about 100 nucleosides in length. They are usually synthesized from nucleoside monomers, but may also be obtained by enzymatic means. Thus, the term “Oligomer” refers to a chain of oligonucleosides which have
  • oligonucleotides includes oligonucleotides, nonionic oligonucleoside alkyl- and aryl-phosphonate analogs, alkyl- and aryl-phosphonothioates, phosphorothioate or phosphorodithioate analogs of oligonucleotides,
  • nucleoside/non-nucleoside polymers are nucleoside/non-nucleoside polymers.
  • the term also includes nucleoside/non-nucleoside polymers wherein one or more of the phosphorus group linkages between monomeric units has been replaced by a non-phosphorous linkage such as a formacetal linkage, a thioformacetal linkage, a sulfamate linkage, a carbamate linkage, an amide linkage, a guanidine linkage, a nitroxide linkage, or a substituted hydrazine linkage.
  • a non-phosphorous linkage such as a formacetal linkage, a thioformacetal linkage, a sulfamate linkage, a carbamate linkage, an amide linkage, a guanidine linkage, a nitroxide linkage, or a substituted hydrazine linkage.
  • nucleoside/non-nucleoside polymers wherein both the sugar and the phosphorous moiety have been replaced or modified such as morpholino base analogs, or polyamide base analogs. It also includes nucleoside/non-nucleoside polymers wherein the base, the sugar, and the phosphate backbone of the non-nucleoside are either replaced by a non-nucleoside moiety or wherein a non-nucleoside moiety is inserted into the nucleoside/non-nucleoside polymer.
  • said non-nucleoside moiety may serve to link other small molecules which may interact with target sequences or alter uptake into target cells.
  • neutral Oligomer refers to Oligomers which have nonionic internucleosidyl linkages between nucleoside monomers (i.e., linkages having no positive or negative ionic charge) and include, for example, Oligomers having internucleosidyl linkages such as alkyl- or aryl- phosphonate linkages, alkyl- or aryl- phosphonothioates, neutral phosphate ester linkages such as phosphotriester linkages, especially neutral
  • a neutral Oligomer may comprise a conjugate between an oligonucleoside or nucleoside/non-nucleoside polymer and a second molecule which comprises a conjugation partner.
  • conjugation partners may comprise intercalators, alkylating agents, binding substances for cell surface receptors, lipophilic agents, nucleic acid modifying groups including photo- cross-linking agents such as psoralen and groups capable of cleaving a targeted portion of a nucleic acid, and the like.
  • Such conjugation partners may further enhance the uptake of the Oligomer, modify the interaction of the Oligomer with the target sequence, or alter the pharmacokinetic distribution of the Oligomer.
  • the essential requirement is that the oligonucleoside or nucleoside/non-nucleoside polymer that the Oligomer conjugate comprises be substantially neutral.
  • substantially neutral in referring to an Oligomer refers to those Oligomers in which at least about 80 percent of the internucleosidyl linkages between the nucleoside monomers are nonionic linkages.
  • acid resistant refers to Oligomers which are resistant, in comparison to deoxyribooligo-nucleotides, to acid-catalyzed depurination by
  • Triplex Oligomer Pair refers to first and second Oligomers which are optionally covalently linked at one or more sites and which are complementary to and are capable of hydrogen bonding to a segment of a single stranded target nucleic acid, such as RNA or DNA, and, thus, together with the single stranded target nucleic acid, are capable of forming a triple helix structure therewith.
  • Third Strand Oligomer refers to Oligomers which are capable of hybridizing to a segment of a double stranded nucleic acid, such as a DNA duplex, an RNA duplex or a DNA-RNA duplex, and forming a triple helix structure therewith.
  • Triplex Oligomer Pair (or first and second Oligomers) or to a Third Strand Oligomer, refers to Oligomers having base sequences which are capable of forming or
  • substantially complementary refers to Oligomers, including Triplex Oligomer Pairs or Third Strand Oligomers which may lack a complement for each nucleoside in the target sequence, have sufficient binding affinity for the target sequence to form a stable duplex or triple helix complex, as the case may be, and thereby specifically recognize the target sequence and selectively inhibit or down-regulate its expression.
  • triplet or "triad” refers a hydrogen bonded complex of the bases of three nucleosides between a base (if single stranded) or bases (if double
  • MRS refers to a methylphosphonothioate
  • internucleosidyl linkages refers to an Oligomer wherein methylphosphonate linkages of Rp chirality alternate with phosphodiester linkages ("DE").
  • internucleosidyl linkages refers to an oligomer wherein methylphosphonate linkages of Rp chirality alternate with phosphorothioate linkages ("PS").
  • internucleosidyl linkages refers to an oligomer wherein methylphosphonothioate linkages of Rp chirality
  • internucleosidyl linkages refers to an oligomer wherein methylphosphonothioate linkages of Rp chirality
  • a "MP(Rp)/DE dimer synthon refers to a dinucleoside wherein the two nucleosides are linked by a
  • nucleosides chirality and one of the nucleosides has a 5'- or 3'-coupling group which when coupled to a 3'-OH or a 5'-OH, of another nucleoside or an oligomer will result in a phosphodiester internucleosidyl linkage.
  • a “MP(Rp)/PS dimer synthon” refers to a dinucleoside wherein the two nucleosides are linked by a
  • methylphosphonate linkage of Rp chirality and one of the nucleosides has a 5'- or 3'- coupling group which when coupled to a 3' -OH or 5'-OH of another nucleoside or an oligomer will result in a phosphorothioate
  • a “MPS(Rp)/DE dimer synthon” refers to a
  • a “MP(Rp)/PS 2 dimer synthon” refers to a dinucleoside wherein the two nucleosides are linked by a
  • methylphosphonate linkage of Rp chirality and one of the nucleosides has a 5'- or 3'- coupling group which when coupled to a 3'-OH or 5'-OH of another nucleoside or an oligomer will result in a phosphorothioate
  • a "2'-O-methyl MP(Rp)/2'-O-methyl DE dimer synthon" refers to a dinucleoside wherein two 2'-O-methyl
  • nucleosides are linked by a methylphosphonate linkage of Rp chirality and one of the nucleosides has a 5'- or 3'- coupling group which when coupled to a 3'-OH or 5'-OH of another nucleoside or an oligomer will result in a phosphodiester internucleosidyl linkage.
  • FIGS 1A and 1B depict representation the
  • Figures 2A and 2B depict phylogenetic analysis of the secondary structure of HPV E7 mRNA around the translation initiation codon.
  • Figure 2A depicts
  • Figures 3A and 3B depict inhibition of cell-free translation of monocistronic E7 in RNA with RNase H mediated cleavers ( Figure 3A) or with steric blockers ( Figure 3B).
  • HPV-11 E7 monocistronic mRNA (circles) or E6/E7 polycistronic mRNA (squares) were translated in rabbit reticulocyte lysates in the absence or in the presence of different concentrations of [MP] [DE] 5 [MP] oligonucleotide 2567-1 [SEQ. ID. NO. 26], ( Figure 3A) or 2'OMeRNA oligonucleotide 2644-1 [SEQ. ID. NO. 18]
  • Figures 4A and 4B depict inhibition of E7 and E6 in cell-free synthesis with RNAse H mediated cleavers:
  • Figure 5 depicts inhibition of transient expression of E7 in COS-7 cells with [Rp-MP/DE] [PS] 5 [Rp-MP/DE] oligomer 3256-1 [SEQ. ID. NO. 32].
  • E7 expression plasmid pcDNAHE7 (5 ug/ml) and different amounts of antisense oligonucleotide were transfected COS-7 cells in the presence of TransfectamTM (Promega). Cells were incubated with transfection mixture for 4 hours, allowed to recover in media plus serum overnight, and labeled with 35 S-cysteine for 5 hours before harvesting.
  • FIGS 6A and 6B depict reduction of E7 mRNA levels in COS-7 cells treated with [Rp-MP/DE] [PS] [Rp-MP/DE] oligomer 3256 [SEQ. ID. NO. 32].
  • E7 expression plasmid pcDNAHE7 (5 ug/ml) alone (lane 1) or together with 5 uM of control oligomer 3215-1 [SEQ. ID. NO. 76] (lane 5) or 0.05 uM (lane 2), 0.5 uM (lane 3) or 5 uM (lane 4) of oligomer 3256-3 [SEQ. ID. NO. 32], was transfected into COS-7 cells using LipofectamineTM (BRL).
  • Figure 7 depicts inhibition of expression of E6 and E7 proteins in cells by chimeric methylphosphonate oligonucleotide 3256.
  • Expression plasmids encoding E6 and E7 were transfected into COS-7 cells together with oligonucleotide 3256-3 [SEQ. ID. NO. 32] at 5 uM. Cells were metabolically radiolabelled the next day and immune precipitates were prepared and analyzed by PAGE and autoradiography as described in Figure 5.
  • Oligonucleotide 3256 targets nucleotides 523-542 of HPV-11, corresponding to the 5' end of the E7 ORF and the 3' end of the E6 ORF.
  • Control oligonucleotide 3218 [SEQ. ID. NO. 46] targets E1.
  • Figure 8 depicts coding potentials of the HPV-11 E-region transcripts.
  • the circular genome is represented in a linear form with the ORFs (open boxes) and their possible functions indicated above. Vertical dashed lines inside each ORF mark the location of the first AUG codon. All viral E-region transcripts are depicted as arrows in the 5'-to-3' direction, with gaps representing introns spliced out of the transcripts and numbers indicating the nucleotide positions of exon boundaries adjacent to splice donors and acceptors.
  • the closed circle at the 5' end of each message represents the proven or putative promoter, and the arrowheads at the 3' ends designate the polyadenylation sites. Coding potentials, as deduced from the cDNA sequences, are drawn as open boxes superimposed on each mRNA arrow, and the encodes proteins are named at the 3' end of each transcript.
  • FIGS 9A to 9C depict structures of E6/E7
  • FIG. 9A depicts E6/E7 mRNA.
  • Figure 9B depicts E6*I/E7 mRNA.
  • Figure 9C depicts E6*II/E7 mRNA.
  • the E6/E7, E6*I/E7 and E6*II/E7 transcripts are depicted as solid lines.
  • the introns resulting from two alternative splicing events within the E6 ORF are depicted as dashed lines, the closed circles at the 5' ends represent the putative promoters.
  • the coding regions for E6, E6*I, E6*II and E7 are indicated by light (E6) or dark (E7) shaded boxes superimposed on the mRNAs .
  • Figure 10 depicts reduction of E7 mRNA levels in HPV transformed CaSki cells treated with [Rp-MP/DE] 6 [PS] 5 [Rp-MP/DE] 9 oligomer 3678 [SEQ. ID. NO. 99].
  • CaSki cells grown in monolayers to about 50% confluency were
  • Figure 11 depicts reduction of E7 mRNA levels in HPV-transformed CaSki cells treated with [Rp-MP/DE] [PS] 5 [Rp-MP/DE] oligomers 3678 [SEQ . ID . NO . 99] , 3679 [SEQ . ID . NO . 100] and 3680 [SEQ . ID . NO . 101] , all them targeted to the translation initiation codon of HPV-16 E7 and with control oligomer 3268 [SEQ. ID. NO. 102].
  • CaSki cells grown in monolayers to about 50% confluency were treated with 1 ⁇ M of oligomers and
  • LipofectamineTM (BRL) for sixteen hours. After the transfection, the cells were washed with CaSki cell culture medium and incubated with the same medium under tissue culture conditions for eight hours. Treatments were repeated for three consecutive days. Intracellular RNA was extracted and RNase protection assays were carried out using an HPV-16 E7 32 P-labeled RNA probe or a GAPDH 32 P-labeled RNA probe. The amount of protected E7 or GAPDH mRNA was quantified using a phosphoimager and the percentage of E7 of GAPDH mRNA respect to
  • Preferred target regions include the portion of an mRNA or pre-mRNA which includes the translation
  • initiation codon a splice donor site, a splice acceptor site, a coding region, a polyadenylation signal, a 3'-untranslated region and a 5'-untranslated region.
  • the mRNA or pre-mRNA codes for a human papilloma virus gene selected from E1, E2, E6 and E7.
  • Preferred target sites include the splice donor at 847 in HPV-6b and HPV-11.
  • a corresponding splice donor site is found in other HPVs, for example, at 880 in HPV-16, at 929 in HPV-18, at 877 in HPV-31, at 894 in HPV-33, at 982 in HPV-5, at 966 in HPV-33, at 982 in HPV-5, at 966 in HPV-8 and at 827 in HPV-1.
  • Figure 8 depicts coding potentials of HPV-11 E-region transcripts and may be used to select appropriate target regions.
  • target regions include those from about - 20 to about +20 nucleosides of a splice donor site or a splice acceptor site, a polyadenylation signal, or within a 3'-untranslated region or a 5'-untranslated region or that about -25 to +35 nucleotides of
  • antisense oligomers which are complementary to a target region of an mRNA or pre-mRNA of an HPV which have from about 14 to about 35 nucleosidyl units, preferably from about 18 to about 24 nucleosidyl units, and more preferably from about 20 to 22 nucleosidyl units.
  • oligomers complementary to a portiotn of the mRNA or pre-mRNA encoding the E7 gene more preferably the target region is in the region of about -25 to about +35 relative to the initiation codon at +1 to +3.
  • the target region is in the region of about -25 to about +35 relative to the initiation codon at +1 to +3.
  • antisense oligomers of the present invention may incorporate any of the variety of
  • internucleosidyl linkages or backbones in certain instances preferred are oligomers have an RNase H-activating region and a non-RNase H-activating region.
  • the RNase H-activating region comprises a segment of at least three consecutive 2'-unsubstituted nucleosides linked by charged internucleosidyl linkages.
  • Preferred charged internucleosidyl linkages include phosphodiester linkages, phosphorodithioate linkages and phosphorothioate linkages.
  • a mixed charged linkage sequence is used which includes at least two different charged nucleosidyl linkages.
  • the non-RNase H-activating region comprises a segment of at least two linked nucleosidyl units linked by internucleosidyl linkages which do not activate (or serve as a substrate for) RNase H. According to an especially preferred aspect, at least one of the
  • Oligomers having an RNase H-activating region are further described in the commonlyassigned and copending United States Patent Application of Lyle J. Arnold, Jr., Mark A. Reynolds and Chris Giachetti, "Chimeric Oligonucleoside Compounds", Serial No. 08/233,778, filed April 26, 1994, 08/238,177, filed May 4, 1994 and PCT/US94/13387, filed November 16, 1994. The disclosures of these applications are incorporated herein by reference.
  • the Oligomers provided herein may form a high affinity complex with a target sequence such as a nucleic acid with a high degree of selectivity.
  • derivatized Oligomers may be used to bind with and then irreversibly modify a target site in a nucleic acid by cross-linking (psoralens) or cleaving one or both strands (EDTA). By careful selection of a target site for cleavage, one of the strands may be used as a molecular scissors to specifically cleave a selected nucleic acid sequence.
  • the Oligomers of the present invention may include an RNase H activating sequence.
  • these antisense Oligomers have a sequence which is complementary to a portion of the RNA transcribed from a selected target gene. Although the exact molecular mechanism of inhibition has not been conclusively determined, it has been suggested to result from
  • duplexes between the antisense Oligomer and the RNA transcribed from the target gene.
  • the duplexes so formed may inhibit translation, processing or
  • interference with or prevention of expression or translation of a selected RNA target sequence may be accomplished by triple helix formation using Oligomers of the present invention as a Triplex Oligomer Pair having sequences selected such that the Oligomers are complementary to and form a triple helix complex with the RNA target sequence and thereby interfere with or prevent expression of the targeted nucleic acid
  • triple strand formation can occur in one of several ways. Basically, two separate or connected Oligomers may form a triple strand with the single stranded RNA. Further descriptions of the use of
  • Oligomers including Triplex Oligomer Pairs
  • a target sequence of double or single stranded nucleic acid by formation of triple helix complexes is described in the copending U.S Patent Applications Serial Nos. 07/388,027, 07/751,813, 07/772,081 and 07/987,746, the disclosures of which are incorporated herein by reference.
  • the Oligomers employed will have a sequence that is complementary to the sequence of the target nucleic acid.
  • absolute is complementary to the sequence of the target nucleic acid.
  • any Oligomer having sufficient complementarity to form a stable duplex (or triple helix complex as the case may be) with the target nucleic acid is considered to be suitable. Since stable duplex formation depends on the sequence and length of the hybridizing Oligomer and the degree of complementarity between the antisense Oligomer and the target sequence, the system can tolerate less fidelity (complementarity) when longer Oligomers are used. This is also true with Oligomers which form triple helix complexes.
  • Oligomers of about 8 to about 40 nucleosidyl units in length which have sufficient complementarity to form a duplex or triple helix structure having a melting temperature of greater than about 40°C under physiological conditions are particularly suitable for use according to the methods of the present invention.
  • Oligomer having methylphosphonate linkages (Second and Third Strands) and one strand of a complementary
  • RNA Oligomer (First Strand) may form a triple helix complex. According to those experiments, the two methylphosphonate strands bind in a parallel
  • triple helix complexes formed by binding a target single stranded RNA and two methylphosphonate Oligomers show high affinity (Tm > 50°C). Formation of these triple helix complexes has been shown to
  • the triple helix complexes can be formed using
  • Oligomers containing naturally occurring bases i.e., A, C, G, T or U.
  • certain stabilizing bases such as 2-amino A (for A) or 5-methyl C may be used in place of the corresponding naturally occurring base.
  • These bases may increase stability of the triple helix complex by having increased hydrogen bonding interactions and stacking interactions with other bases. Increased stability may result in increased affinity constants which increase potency.
  • the Oligomers provided herein may be derivatized to incorporate a nucleic acid reacting or modifying group which can be caused to react with a nucleic acid segment or a target sequence thereof to irreversibly modify, degrade or destroy the nucleic acid and thus
  • Oligomers may be used to inactivate or inhibit or alter expression of a particular gene or target sequence of the HPV in a living cell, allowing selective inactivation or inhibition or alteration of expression.
  • the target sequence may be DNA or RNA, such as a pre-mRNA or an mRNA.
  • mRNA target sequences include an initiation codon region, a coding region, a
  • Antisense therapy as used herein is a generic term which includes the use of specific binding Oligomers to inactivate undesirable DNA or RNA sequences in vitro or in vivo.
  • Antisense therapy includes targeting a specific DNA or RNA target sequence through complementarity or through any other specific binding means, in the case of the present invention by formation of duplexes or triple helix complexes.
  • the Oligomers for use in the instant invention may be administered singly, or combinations of Oligomers may be administered for adjacent or distant targets or for combined effects of antisense mechanisms with the foregoing general mechanisms.
  • the Oligomers can be formulated for a variety of modes of administration, including oral, topical or localized administration. It may be beneficial to have pharmaceutical formulations containing acid resistant Oligomers that may come in contact with acid conditions during their manufacture or when such formulations may themselves be made acidic, to some extent, in order to more compatible with the conditions prevailing at the site of application, e.g., the acid mantle of the skin. Techniques and
  • the Oligomer active ingredient is generally combined with a carrier such as a diluent or excipient which may include fillers, extenders, binding, wetting agents, disintegrants, surface-active agents, erodible polymers or lubricants, depending on the nature of the mode of administration and dosage forms.
  • a carrier such as a diluent or excipient which may include fillers, extenders, binding, wetting agents, disintegrants, surface-active agents, erodible polymers or lubricants, depending on the nature of the mode of administration and dosage forms.
  • Typical dosage forms include tablets, powders, liquid
  • preparations including suspensions, emulsions and solutions, granules, and capsules.
  • Oligomers of the present invention may be particularly suited for oral administration which may require exposure of the drug to acidic conditions in the stomach for up to about 4 hours under conventional drug delivery conditions and for up to about 12 hours when delivered in a sustained release from.
  • the Oligomers having 2'-O-alkyl nucleosidyl units may be particularly suited for formulation in
  • the Oligomers for use in the invention are formulated into ointments, salves, eye drops, gels, or creams, as is generally known in the art.
  • Systemic administration can also be by transmucosal or transdermal means, or the compounds can be
  • penetrants appropriate to the barrier to be permeated are used in the formulation.
  • penetrants are generally known in the art, and include, for example, bile salts and fusidic acid derivatives for transmucusal administration.
  • detergents may be used to facilitate permeation.
  • Transmucosal administration may be through use of nasal sprays, for example, as well as formulations suitable for
  • This dry solid foam was then dissolved in 500 ml of acetonitrile ("ACN”), and with manual stirring, treated all at once with 404 ml tetrazole (180 mM, 0.45 M tetrazole in THF). Manual stirring is continued for 30 seconds and then the flask is allowed to stand for another 2.5 minutes, after which time the reaction mix is treated all at once with 275 ml of an oxidizer solution (I 2 /H 2 O/lutidine/THF; 25 g/2.5 ml/100 ml/900 ml). The solution was stirred manually and allowed to stand at room temperature for 15 minutes.
  • ACN acetonitrile
  • the crude dimer was run on HPLC (reverse phase, Waters C18 bondapak) with a program (ACNMETH) starting with 50% acetonitrile and 0.1 M triethylammonium acetate (TEAA, pH ⁇ 7.0) which increased to 100% acetonitrile over 20 minutes with a linear gradient. Two major peaks were resolved, one at 4.5 minutes, which is residual lutidine and the other at 14.5 minutes which is the mixture of R p and S p diastereomers.
  • HPLC reverse phase, Waters C18 bondapak
  • ACNMETH triethylammonium acetate
  • the ratio of R p and S p was determined quantitatively by taking a 5 mg aliquot of the crude product and dissolving it in 1.5 ml of acetonitrile along with 0.5 ml of tetrabutylammonium fluoride (TBAF, 1 M solution in THF). After standing at room temperature for 10 minutes the sample was run on HPLC . Two new peaks were observed at 6.5 and 7.1 minutes and the later eluting peak was gone. The first new peak, which is believed to be the S p diastereomer, represented 66% (2/1) of the normalized value for the two peaks.
  • the crude product was also analyzed by the (normal phase silica plate) in 75/25 EtOAc/CH 2 Cl 2
  • the R p diastereomer was separated on normal phase silica using a methanol step gradient in 75/25
  • the CT-3'-OH dimer 5.5 g (6 mM), prepared as described in part A above, was rendered anhydrous with two co-evaporations with pyridine.
  • the resulting solid foam was released from the rotary evaporator with argon and stoppered with a rubber septa.
  • the solid foam was dissolved in 100 ml of 9/1, ACN/CH 2 Cl 2 , then treated with 1.7 ml triethylamine (TEA, 12 mM). With magnetic stirring, the reaction mix was treated dropwise at room temperature with 1.5 ml chloromethyl-N,N-diisopropylamino phosphine (Cl-MAP, 8 mM).
  • the reaction was monitored on HPLC (ACNMETH) and after 1.5 hours was complete, showing two main products, one at 3.5 minutes which was pyridine and a second at 14.3 minutes which was the desired amidite.
  • the reaction mixture was concentrated on a rotary evaporator using a partial vacuum; the flask which contained the resulting light amber sludge was released under argon and capped.
  • the crude product was
  • reaction mixture (reaction was determined to be complete by HPLC) was concentrated to dryness. The residue was dissolved in 20 ml ethyl acetate/heptane (1:1) with 4% TEA, then loaded onto 40 g silica gel equilibrated with the same solvent system. All UV absorbing eluent from the column was collected and pooled, then concentrated to give 5.5 g of the above-identified product (yield about 90%).
  • the solid foam was then dissolved in 70 ml methylene chloride and treated (with rapid magnetic stirring) all at once with 70 ml benzene sulfonic acid, 2% by weight in 2:1 methylene chloride/methanol. After stirring for 15 minutes at room temperature, the reaction mixture was quenched with 10 ml TEA. The resulting detritlylated compound was stripped down to a thick amber oil which was then loaded onto 150 g. silica gel equilibrated in heat methylene chloride. The product was eluted from the column using 2% methanol (in methylene chloride). After drying, 3.51 g of the above identified product were obtained (yield about 80%).
  • Example 2D it was rendered anhydrous by 1 X 5 ml co-evaporation with acetonitrile. The dry foam was then released from the vacuum system under argon gas,
  • dimer synthons are prepared by following the procedures described in Example 2, except that in
  • Paragraph C an equivalent amount of 3H-1,2-benzodithiole-3-one, 1,1-dioxide (Beaucage reagent) is substituted for cumene hydroperoxide.
  • eaucage reagent 3H-1,2-benzodithiole-3-one, 1,1-dioxide
  • dimer synthons are prepared by following the procedures of Example 1, except in Paragraph A, an equivalent amount 3-H-1,2-benzodithiole-3-one, 1,1-dioxide (Beaucage reagent) is substituted for the oxidizer solution (I 2 /H 2 O/lutidine/THF).
  • the MP(R p )/PS2 dimer synthons are prepared as follows. Isometrically pure R p dinucleosides having a free 3'-OH are prepared according to the methods
  • Example 1B The product is worked up and purified using the procedures of Example 1B for isolation of the MP(R p )/DE phosphoramidite.
  • the MPS (R p ) /PS2 dimer synthons are prepared as follows.
  • the isometrically pure R p dinucleoside with a free 3'-OH is prepared according to the methods of
  • Example 4 Using the dinucleoside, the dimer synthon is prepared by the methods of Example 5.
  • the protected dinucleoside methylphosphonamidite (22 mg each per required coupling) freshly co-evaporated with pyridine and toluene to ensure dryness were placed into dried 1 ml glass autosampler vials and dissolved in anhydrous acetonitrile to a concentration of 0.1 M (200 ⁇ l per coupling).
  • the vessels were purged with argon and tightly sealed with screw caps with teflon septa.
  • a 1 ⁇ mole scale DNA synthesis column (Milligen) was filled with 1 ⁇ mole of methacrylate support bound deoxyadenosine.
  • the column was attached to a ring stand in a vertical orientation.
  • a male-male luer fitting was attached to the bottom along with an 18 gauge needle to control the effluent.
  • the column was washed with 10 ml acetonitrile using a syringe.
  • nucleoside was detritylated by passing 3 ml of 2% dichloroacetic acid in dichloromethane through the column over 1.5 minutes. The orange, dimethoxytrityl cation bearing solution was reserved. The column was washed twice with 10 ml each of anhydrous acetonitrile.
  • the first coupling was accomplished as follows: 10 ml more anhydrous acetonitrile was passed through the column. Then, 200 ⁇ l of the CT methylphosphonamidite was drawn into a 1 ml syringe. Next, 200 ⁇ l of 0.45 M tetrazole in anhydrous acetonitrile was likewise drawn into the syringe containing the methylphosphonamidite. The reagents were rapidly mixed in the syringe, then slowly passed through the column dropwise over three minutes, being sure to lightly draw the plunger up and down to ensure adequate mixing with the support.
  • the oligomer was then cleaved from the support and deprotected.
  • the support bound oligomer was removed from the synthesis cartridge and placed in a glass 1 dram vial with a screw top.
  • the support was treated for 30 minutes at room temperature with 1 ml of a solution of acetonitrile/ethanol/NH 4 OH (9/9/1). Then, 1 ml of ethylenediamine was added to the reaction vessel and the reaction allowed to sit for 6 hours at ambient
  • the supernatant containing the oligomer was then removed from the support and the support was rinsed twice with 2 ml of 1/1 acetonitrile/water; the washings were combined with the supernatant.
  • the combined solution was diluted to 30 ml total volume with water and neutralized with approximately 4 ml of 6 N HCL.
  • the neutralized solution was desalted using a Waters C-18 Sep-Pak cartridge which was pre-equilibrated with 10 ml acetonitrile, 10 ml of 50% acetonitrile/100 mM triethylammonium bicarbonate, and 10 ml of 25 mM triethylammonium bicarbonate, sequentially. After the reaction solution was passed through the column, it was washed with 30 ml of water. The product was then eluted with 5 ml of 1/1
  • the oligomer was purified on HPLC using a Beckman Ultrasphere-reverse phase 4.5 X 250 mm column with an increasing gradient of acetonitrile in 0.5 M
  • the above-identified oligomer can be synthesized on an automated DNA synthesizer.
  • the appropriate dimer synthons (as used above in the manual synthesis) are dissolved in acetonitrile to a concentration of 0.1 M as described above.
  • the amidite solutions are placed in conical vessels on a Millipore Expedite DNA Synthesizer. All other reagents (oxidizer, deblock, capping reagents and activator) are prepared as described above for the manual synthesis, and applied to the appropriate positions on the instrument as
  • An oligomer having the sequence 5'(C*U)-(C*U)-(C*U)- (C*U)-(C*U)-(C*U)-A-3' was prepared using 2'-O-methyl MP(R p )/2'-O-methyl DE dimer synthons prepared according to Example 2 hereinabove.
  • dimer synthons were dissolved in acetonitrile to a concentration of 0.1 M. All other reagents used were as described in Example 8.
  • a 1 ⁇ mole scale DNA synthesis column (Millipore) was filled with 1 ⁇ mole of methacrylate support bound deoxyadenosine. The dimer synthons were coupled
  • Example 8 except that the coupling time was extended to two minutes.
  • the overall coupling efficiency based on dimethoxytrityl absorbance was 50%, for an average of 91% per coupling.
  • the dimethoxytrityl group was removed from the oligomer at the end of the synthesis.
  • the deprotection was carried out as described in Example 8.
  • the crude yield was 103 OD 260 units.
  • the oligomer was purified on HPLC with a Beckman
  • This oligomer can also be synthesized on an
  • the deprotection is carried out as described in Example 8.
  • the oligomer can be purified on HPLC using as described above for the manual synthesis.
  • the grouped dinucleosides indicate coupled dimers and the asterisk indicates where the stereochemistry is fixed (chirally defined or chirally pure) as the fast eluting isomer on silica gel (identified as R p ).
  • methylphosphonamidites prepared using methods such as those described in Examples 1A and 1C above. Manual couplings were used to synthesize the oligomer to conserve reagent, although the process can be done on an automated DNA synthesizer from the 3' terminus starting with support-bound cytidine.
  • a 1 ⁇ mole scale Milligen DNA synthesis column was filled with 1 ⁇ mole of support bound cytidine.
  • the column was attached to a ring stand in a vertical orientation.
  • a male-male leur fitting was attached to the bottom along with an 18 gauge needle to control the effluent.
  • the column was washed with 10 ml of ACN using a syringe.
  • the support bound nucleoside was then detritylated by passing 3 ml of 2% dichloroacetic acid in dichloromethane through the column over 1.5 minutes. The orange, dimethoxytrityl cation bearing solution was reserved.
  • the column was washed twice with 10 ml each of ACN (anhydrous).
  • the first coupling was accomplished by passing 10 ml more ACN (anhydrous) through the column. Then, 200 ⁇ l of the TG methylphosphonamidite was drawn into a 1 ml syringe. Next, 200 ⁇ L of 0.45 M tetrazole in anhydrous ACN was likewise drawn into the syringe containing the methylphosphonamidite. The reagents were rapidly mixed in the syringe, then slowly passed through the column dropwise over 3 minutes, being sure to lightly draw the plunger up and down to ensure adequate mixing with the support.
  • the synthetic cycle was then repeated with each dinucleotide methylphosphonamidite until the synthesis was completed.
  • the order of addition of dimers after the initial T*G coupling was C*C, C*T, A*G, T*T, C*C, T*T, G*C, and T*A.
  • the dimethoxytrityl group was removed from the oligomer at the end of the synthesis.
  • the oligomer was then cleaved from the support and deprotected.
  • the support bound oligomer was removed from the synthesis cartridge and placed in a glass 1 dram vial with a screw top.
  • the support was treated for 30 minutes at room temperature with 1 ml of a solution of acetonitrile/ethanol/NH 4 OH (9/9/1).
  • 1 ml of ethylenediamine was added to the reaction vessel and the reaction mixture allowed to sit for 6 hours at ambient temperature in order to go to completion.
  • the supernatant containing the oligomer was then removed from the support and the support was rinsed twice with 1 ml of 1/1 acetonitrile/water; the washings were combined with the supernatant.
  • the combined solution was diluted to 50 ml total volume with water and neutralized with approximately 1.7 ml of glacial acetic acid.
  • the neutralized solution was desalted using a Waters C-18 Sep-Pak cartridge which was pre-equilibrated with 5 ml acetonitrile, 5 ml of 50% acetonitrile/water, and 5 ml of water, sequentially. After the reaction solution was passed through the column, it was washed with 50 ml of water. The product was then eluted with 2 ml of 1/1 acetonitrile/water.
  • the oligomer was purified by HPLC on a reverse phase column (Poros II R/H 4.6 ⁇ 100 mm) using a gradient of acetonitrile in water.
  • the grouped dinucleotides indicate coupled dimers and the asterisk indicates where the stereochemistry is fixed.
  • a 1 ⁇ mole scale Milligen DNA synthesis column was filled with 1 ⁇ mole of methacrylate support bound 2'-deoxycytidine.
  • the column was attached to a ring stand in a vertical orientation.
  • a male-male luer fitting was attached to the bottom along with an 18 gauge needle to control the effluent.
  • the column was washed with 10 ml of ACN using a syringe.
  • the support bound nucleoside was then detritylated by passing 3 ml of 2.5%
  • the reagents were rapidly mixed in the syringe, then slowly passed through the column dropwise over 1 minute, being sure to lightly draw the plunger up and down to ensure adequate mixing with the support. After 3 minutes, 1 ml of the oxidizing reagent (0.1 M I 2 in 74.25% THF, 25% 2, 6-lutidine, and 0.25% water) was passed through the column over 1 minute. The column was then washed with 20 ml of ACN. The column was then treated for 1 minute with 600 ⁇ l of a solution containing 20% (v/v) acetic anhydride, 30% (v/v) ACN, 50% (v/v) pyridine, and 0.312% (w/v)
  • the synthetic cycle was then repeated with each dinucleotide methylphosphonamidite until the synthesis was completed.
  • the order of addition of dimers after the initial G*T coupling was T*T, T*G, C*A, T*G, C*A, T*C, C*T and G*T.
  • the dimethoxytrityl group was removed from the oligomer at the end of the synthesis.
  • the oligomer was then cleaved from the support and deprotected.
  • the support bound oligomer was removed from the synthesis cartridge and placed in a glass 1 dram vial with a screw top.
  • the support was treated for 30 minutes at room temperature with 1 ml of a solution of acetonitrile/ethanol/NH 4 OH (9/9/1).
  • 1 ml of ethylenediamine was added to the reaction vessel and the reaction allowed 6 hours to go to completion.
  • the supernatant containing the oligomer was then removed from the support and the support was rinsed twice with 1 ml of 1/1 acetonitrile/water; the washings were combined with the supernatant.
  • the combined solution was diluted to 30 ml total volume with water and neutralized with approximately 1.7 ml of glacial acetic acid.
  • the neutralized solution was desalted using a Waters C-18 Sep-Pak cartridge which was pre-equilibrated with 5 ml acetonitrile, 5 ml of 50% acetonitrile/water, and 5 ml of water, sequentially. After the reaction solution was passed through the column it was washed with 5 ml of water. The product was then eluted with 2 ml of 1/1 acetonitrile/water.
  • the oligomer was purified by HPLC on a reverse phase column (Poros II R/H 4.6 ⁇ 100 mm) using a gradient of acetonitrile in water.
  • the grouped dinucleosides indicate the coupled dimers and the asterisks indicates where the
  • This oligomer was prepared using automated synthesis coupling G*A, G*G and A*G MP (R p )/MP dimer synthons prepared according to the procedures of Examples 1A and
  • Synthesizer which as equipped with end-line filters to remove particulates. All other reagents (oxidizer, deblock, capping reagents and activator) were prepared and applied to the appropriate positions on the
  • a 1 ⁇ mole scale DNA synthesis column (Millipore) was filled with 1 ⁇ mol of methacrylate support-bound
  • deoxyguanosine was placed on the DNA synthesizer.
  • the dimers were coupled sequentially from the 3' terminus.
  • the dimethoxytrityl protecting group was removed from the oligomer at the end of the synthesis.
  • the oligomer was then cleaved from the support and deprotected.
  • the support bound oligomer was removed from the synthesis cartridge and placed in a glass 1 dram vial with a screw top.
  • the support was treated for 30 minutes at room temperature with 1 ml of a solution of acetonitrile/ethanol/NH 4 OH (9/9/1).
  • 1 ml of ethylenediamine was added to the reaction vessel and the reaction allowed 6 hours to go to completion.
  • the supernatant containing the oligomer was then removed from the support and the support rinsed twice with 1 ml of 1/1 acetonitrile/water, when combined with the supernatant.
  • the combined solution was diluted to 50 ml total volume with water and neutralized with
  • the crude yield was 87 OD 260 units.
  • the Oligomers was purified on HPLC using a /S-cyclobond standard phase 4.5 X 250 mm column (Azetec, Inc. Whippany, NJ) with a decreasing gradient (80% to 40%) of acetonitrile in 0.05 M triethylammonium acetate (pH 7).
  • the isolated yield was 22 OD 260 units (25%).
  • the product was characterized by electron spray mass spectrometry (calc. 5407/found 5401).
  • internucleosidyl linkages is prepared using dimer synthons. All the parameters of the synthesis,
  • Example 8 except that the oxidizing reagent is replaced by a 0.1 M solution of 3H-1,2-benzodithiole-3-one, 1,1-dioxide or a 0.1 M solution of sulfur in 1/1 carbon disulfide/diisopropylethylamine.
  • Example 8 except that the oxidizing reagent is replaced by a 0.1 M solution of 3H-1,2-benzodithiole-3-one, 1,1-dioxide or a 0.1 M solution of sulfur in 1/1 carbon disulfide/diisopropylethylamine.
  • the preparation of an oligomer having alternating MP(R p )/MPS internucleosidyl linkages is accomplished using dimer synthons prepared according to Examples 1A and 1C and dissolved and stored over molecular sieves.
  • the oxidizing reagent is a 0.1 M solution of 3H-1,2-benzodithiole-3-one, 1,1-dioxide ("Beaucage Reagent", See, Iyer, R.P. et al., JACS 112:1254-1255 (1990)) or a 0.1 M solution of sulfur in 1/1 carbon disulfide/ diisopropylethylamine, with synthesis proceeding
  • This oligomer is prepared using the dimer synthons as described in Examples 2A-2D and 2F and following the general synthetic procedures of Example 8 of U.S. Patent Application Serial No. 08/154,013, except that the oxidizing reagent described therein is a 0.1M solution of 3H-1,2-benzodithiole-3-one, 1,1-dioxide or a 0.1 M solution on 1/1 carbon disulfide/diisopropylamine.
  • This oligomer is prepared using dimer synthons as described in Example 3 above and by following the parameters of synthesis, deprotection and purification of Example 19.
  • This oligomer is prepared using dimer synthons prepared according to Examples 1A and 1C, substituting Beaucage reagent for the oxidizer in Example 1A, and by following the parameters of synthesis, deprotection and purification as described above in Example 12.
  • This oligomer is prepared using dimer synthons as referred to in Example 21 and by following the
  • Dimer synthons useful in the preparation of the oligomers of the present invention may be prepared using 2'-fluoronucleosides. Methods for preparation of 2'-fluoronucleosides have been reported and are known to those skilled in the art. (See, e.g.: Codington, JOC Vol. 29 (1964) (2'-F U); Mangel, Angew. Chem. 16:557-558 (1978) and Doen, JOC 11:1462-1471 (1967) (2'-F C);
  • the preparation of dimer synthons using 2'-fluoronucleosides may be accomplishing using the
  • Example 24 See, e.g., Examples 2, 3, and 7).
  • the resulting dimer synthons may be used to prepare oligomers using methods analogous to the methods used for the 2'-O-methyl dimer synthons such as in Example 9.
  • Example 24
  • trimer synthons are prepared using the MP(R p )/MP dimer synthons of Example 1C.
  • the dimer synthon is coupled to a 5'-hydroxy, 3'-silylated nucleoside according to the methods of Example 1A for the coupling of the 3'-nucleoside to the monomer
  • methylphosphonamide (1.25 equivalents) are weighed into a round bottom flask and dried by co-evaporation with acetonitrile. The resulting foam is dissolved in acetonitrile and treated with a solution of 0.45 M tetrazole in acetonitrile (4.5 equivalents). After 3 minutes, the reaction mixture is oxidized and the reaction product is worked up as described in Example 1A. The diastereoisomers of the 3'-silylated trimer are resolved on a silica gel column as described in Example 1A for resolution of the dimer isomers.
  • the diastereoisomers of the 3'-silylated trimer are resolved on a silica gel column as described in Example 1A for resolution of the dimer isomers.
  • internucleosidyl linkages is converted to a trimer synthon by reaction with chloro- ⁇ -cyanoethoxy-N,N- diisopropylaminophosphoramidite using methods as
  • trimer synthon is worked up and purified using methods as described in Example IB to achieve the MP (R p )/MP (R p ) /DE trimer.
  • an MP (R p )/MP(R p )/MP phosphoramidite synthon may be obtained by using
  • Examples 1 and 24 can be prepared using 2'-O-allyl nucleosides.
  • synthons using procedures described hereinabove.
  • the synthons are used to prepare oligomers using methods such as those described in Examples 10, 11, 12, 13 and others above.
  • Oligoribonucleotides used in the present examples may be synthesized using general procedures such as described below.
  • oligonucleotides were handled under sterile, RNase-free conditions. Water was
  • the oligonucleotides were deprotected and cleaved from the support by first treating the support bound oligomer with 3/1 ammonium hydroxide/ethanol for 15 hours at 55°C. The supernatant, which contained the oligonucleotide, was then decanted and evaporated to dryness. The resultant residue was then treated with 0.6 mL of 1 M tetrabutylammonium fluoride in
  • oligoribonucleotides Purification of the oligoribonucleotides was carried out by polyacrylamide gel electrophoresis (PAGE) containing 15% 19/1 polyacrylamide/bis-acrylamide and 7 M urea using standard procedures (See Maniatis, T. et al., Molecular Cloning: A Laboratory Manual, pages 184-185 (Cold Spring Harbor 1982)). The gels were 20 cm wide by 40 cm long and 6 mm in width. The
  • oligoribonucleotides (60 OD Units) were dissolved in 200 ⁇ L of water containing 1.25% bromophenol blue and loaded onto the gel. The gels were run overnight at 300 V. The product bands were visualized by UV backshadowing and excised, and the product eluted with 0.5 M sodium acetate overnight. The product was desalted with a Waters C18 Sep-Pak cartridge using the manufacturer supplied protocol. The product was then 32 P labelled by kinasing and analyzed by PAGE.
  • OXIDIZER reagent 25 g/L iodine in 0.25% water, 25% 2,6-lutidine, 72.5% tetrahydrofuran.
  • CAP A 10% acetic anhydride in acetonitrile.
  • CAP B 0.625% N,N-dimethylaminopyridine in pyridine.
  • the dimethoxytrityl group was removed from the oligonucleotide at the end of the synthesis.
  • the oligonucleotide was then cleaved from the support and deprotected.
  • oligonucleotide was removed from the synthesis cartridge and placed in a glass 1 dram vial with a screw top.
  • the support was treated for 30 minutes at room temperature with 1 ml of a solution of acetonitrile/ethanol/NH 4 OH (9/9/1). Then, 1 ml of ethylenediamine was added to the reaction vessel and the reaction allowed 6 hours to go to completion.
  • oligonucleotide was then removed from the support and the support rinsed twice with 2 ml of 1/1
  • acetonitrile/water when combined with the supernatant.
  • the combined solution was diluted to 30 ml total volume with water and neutralized with approximately 4 ml of 6 N HCl.
  • the neutralized solution was desalted using a Waters C-18 Sep-Pak cartridge which was pre-equilibrated with 10 ml acetonitrile, 10 ml of 50% acetonitrile/100 mM triethylammonium bicarbonate, and 10 ml of 25 mM triethylammonium bicarbonate, sequentially. After the reaction solution was passed through the column it was washed with 30 ml of water. The product was then eluted with 5 ml of 1/1 acetonitrile/water.
  • the oligonucleotide was purified by HPLC on a reverse phase column (Whatman RAC II) using a gradient of acetonitrile in 50 mM triethylammonium acetate.
  • MP(R p )/MP dimer synthons contained a
  • R p -MP/DE dimer synthons contained a ⁇ -cyanoethyl phosphoramidite coupling group at the 3'-end. Both types of dimer synthons were synthesized as described in Example 1. Methylphosphonamidite monomer synthons were synthesized at JBL Scientific (San Luis Obispo, CA). Betacyanoethyl phosphoramidite monomer synthons were purchased from Milligen/Biosearch.
  • An expression vector having an insert coding for HPV11 E6/E7 was prepared using the expression vector pRc/CMV (Invitrogen) as follows:
  • the plasmid pRC/CMV was linearized with Hind III.
  • the recessed 3' ends were filled with the 5 '-3'
  • the vector and insert were ligated with T 4 DNA ligase and transformed into DH5 ⁇ E. Coli . recombinants were screened for appropriate insert and orientation as well as E6/E7 transcription and translation activity.
  • This plasmid (pRc/CMV11-E6/E7) was used to prepare the polycistronic mRNA used in the cell free translation system described in Example F.
  • An expression vector having an HPV-11 E7 insert was prepared using pcDNA-1 (Invitrogen) as follows.
  • the plasmid pcDNA was digested with BamH I and with Xba I.
  • a fragment containing the complete open reading frame of HPV-11 (from -30 till the termination codon) flanked by Bam HI and Xba I restriction sites was prepared by PCR using standard protocols.
  • the digested vector and fragment were ligated with T4 DNA ligase and transformed into MC 1061/P3 cells. Recombinants were screened for appropriate insert, transcription and translation.
  • This plasmid (pcDNA E7) was used to prepare the monocistronic mRNA used in the cell-free translation system described in Example G and in the transient expression assay in COS-7 cells described in Example J and in the RNase H cleavage assay of Example I.
  • An expression vector having an HPV-11 E1 insert was prepared using the expression vector pRc/CMV
  • the plasmid pRC/CMV was linearized with Hind III.
  • the recessed 3' ends were filled in with T 4 DNA
  • a full length clone of HPV-11 cloned at the Bam HI site in pBR322 was digested with the restriction enzyme ApaL I.
  • the recessed 3'-ends were filled in with the 5' -3' polymerase activity of the Klenow fragment of DNA polymerase I.
  • the modified DNA was next cut with Spe I and a 2428 base pair fragment containing the complete E1 ORF was agarose gel purified.
  • the modified vector and E1 insert were ligated with T 4 DNA ligase and transformed into DH5 ⁇ . E. Coli . Recombinants were screened for appropriate insert, transcription and translation.
  • An expression vector having an HPV-11 E2 insert was prepared using pRc/CMV (Invitrogen) as follows.
  • the plasmid pRC/CMV was linearized with Hind III, followed by treatment with calf thymus alkaline
  • the modified vector and E2 insert were ligated with T 4 DNA ligase and transformed into DH5 ⁇ E. Coli .
  • This plasmid (pRc/CMVII-E2) was used in the cell-free translation system of Example 0.
  • Figures 2A and 2B explain the pseudoknot structures for HPV-6b and HPV-11. Please note that the AUG in boldface is the start codon for the E7 mRNA. The pseudoknot starts 6 nucleotides upstream from the from the HPV-11 E7 AUG. In the case of high risk HPVs a variable number of nucleotides is present between the pseudoknot structure and the initiation codon of E7.
  • CAT chloramphenicol acetyl transferase
  • RNAseH and oligonucleotide at either 0.01, 0.1 or 1 ⁇ M.
  • CAT mRNA was co-translated as negative control. Please note that all the oligonucleotides tested, but 2589-1 and 2590-1, and 2492-1 are fully complementary to HPV-11 as well as the HPV-6b sequence.
  • oligonucleotide 2498-1 complements HPV-6b as well as HPV-11 and showed good specific translation inhibition of E7, it was selected as our preferable oligomer sequence.
  • Example E since the phosphodiester data shows a decrease in oligomer activity as the oligomer overlaps with the pseudoknot.
  • HPV-11 E7 may be translated from a
  • oligonucleotide 2644-1 [SEQ. ID. NO. 18], a 2'-OMeRNA, was used as an example of a steric blocker.
  • oligonucleotide 2567-1 was able to inhibit E7 translation whether E7 was translated from the monocistronic (circles) or from the polycistronic mRNA (squares).
  • the oligomer was only slightly more active on the monocistronic mRNA than on the
  • FIG. 3B depicts the results obtained with oligonucleotide 2644-1. This result showed that the activity of the steric blocker on either mRNA was lower than the activity observed with the RNase H mediated cleaver 2567-1, and that oligomer 2644-1 produced very little inhibition of E7 synthesis when it is translated from the polycistronic mRNA. In the case of both the oligomers, no effects on CAT translation were observed, indicating that their was activity was very specific.
  • Tm melting temperatures
  • oligomer and synthetic RNA target in 1 ml of buffer containing 20 mM DPO 4 , pH 7.2, 0.1 mM NaCl, 0.1 mM EDTA and 0.03% sarkosyl.
  • the reaction mixtures were heated by 80°C and then slowly cooled to 4°C over approximately 4 to 6 hours. The annealed samples were then transferred to 1 cm quartz cuvettes; absorbance at 260 nm as a function of
  • the temperature was varied from 5°C to 80°C at a ramp rate of 1°C/minute.
  • the Tm for each melt profile was defined at the point
  • RNA polymerase Ambion MegaScript
  • the E7 RNA was incubated at a concentration of 100 nM in the presence of 0.04 units ⁇ L E. Coli . RNAse H (Promega), 3.5 mM MgCl 2 , 25 mM KCl, 70 mM NaCl and 20 mM potassium acetate at 37°C for 30 minutes. Reactions were stopped by addition of formamide gel loading buffer followed by heating to 100°C for 5 minutes.
  • COS-7 cells obtained from ATCC [Catalog CRL 1651] were seeded at 1 X 10 5 cells/well in 24 well plates and then cultured overnight in cell culture media (90% DMEM, 10% fetal bovine serum and 50 I.U./ml penicillin, 50 mg/ml streptomycin and 0.25 ⁇ g/ml amphotericin B).
  • a transfection cocktail of 2.5 ⁇ g/mL pcDNA1E7, 50 ⁇ g/mL transfeetam (Promega) and varying concentrations of oligomer was prepared and incubated for 15 minutes at room temperature after a 2 second vortex mix.
  • RIPA buffer (10 mM Tris-Cl [pH 7.4], 150 mM NaCl, 1% Triton X-100, 0.1% sodium dodecyl sulfate, 0.5% sodium deoxycholate) and combined with sample buffer lysate.
  • E7 synthesis was evaluated by immunoprecipitation of E7 protein with goat anti-HPV-11 E7 serum and protein A sepharose beads (Sigma). Immunoprecipitated E7 protein was quantitated by SDS-PAGE and phospho-image analysis. Total protein synthesis was evaluated by SDS-PAGE and phospho-image analysis of a fraction of the transfected cell lysate before immunoprecipitation.
  • oligonucleotides 3214-1, 3257-1 and 3256-1 which contain all phosphorothioate ([PS]) or alternating phosphorothioate/phosphodiester ([PS/DE]) linkages in the middle and chiral methylphosphonate dimers linked by phosphodiester linkages ([Rp-MP/DE]) as end-blocks are potent inhibitors of transient expression of HPV E7 protein in COS-7 cells.
  • oligonucleotides 3327-1 and 3336-1 were less potent than the corresponding chimeras with [Rp-MP/DE] ends.
  • the intracellular levels of E7 mRNA after treatment of transient transfected cells with a chimeric oligomer were determined using a RNase protection assay.
  • the RNA probe used in the assay complements HPV-11 N564 to N841, so that if E7 mRNA is present, a protected band of 278 nucleotides should be expected.
  • As control for the RNase protection assay we determined that the
  • the phosphoimage presented in Figure 6A shows that treatment of the cells with oligomer 3256 [SEQ. ID. NO. 32] at 0.05, 0.5 or 5 uM produced a dose-dependent decrease in the amount of E7 probe (lanes 1-4),
  • HPV-11 E6 proteins are translated from the polycistronic E6/E7 mRNA containing the ORF of E6 upstream from the ORF of E7. Since oligomers targeted to the translation initiation codon of E7 will also complement the 3' portion of the ORF of E6, it was important to determine the activity of these oligomers on E6 production.
  • HPV-11 E6/E7 polycistronic mRNA was translated in rabbit reticulocyte lysates in the absence or in the presence of different concentrations of oligomers.
  • Figures 4A and 4B depict the results obtained with phosphodiester oligonucleotide 2498-1, [SEQ. ID. NO. 14] ( Figure 4A) and with [MP] [DE] 5 [MP] oligomer 2567-1, [SEQ. ID. NO. 26] ( Figure 4B).
  • HPV-11 E6 is translated from a E6/E7 mRNA containing the E6 ORF upstream from the E7 ORF ( Figure 1).
  • oligonucleotides that are targeted to the translation initiation codon of E7 will also complement the 3' portion of the E6 ORF.
  • E6 plasmids pcDNAHE7
  • pED/E6 contains the HPV-11 E6 gene plus 32 upstream bases (HPV
  • nucleotides 72-555 behind the adenovirus major late promoter and tripartite leader], encoding either E7 or E6, were transfected into COS-7 cells together with chimeric oligomer 3256 [SEQ. ID. NO. 32] at 5 uM.
  • E6 and E7 protein were evaluated after 18 hours by metabolic labeling and precipitation with specific anti-E7 or anti-E6 serum, followed by
  • oligomer 3256 inhibited expression of E7 (lane f) as expected. This oligonucleotide also blocked expression of E6 (lane c).
  • a control chimeric oligomer 3218 targeted to the translation start site of the E1 protein of HPV-11 had no effect on expression of E7 (lane g) and little effect on E6 (lane d).
  • chimeric oligonucleotides are effective when targeted to either the translation start site of an mRNA (E7 in this case) or to the 3' end of an ORF (E6). This results supports the idea that chimeric oligonucleotides can block synthesis of multiple
  • VERO cells approximately 2 X 10 5 cells/ml
  • Plasmid DNA was diluted in
  • PBS to a concentration of 20 ng/ ⁇ l (E7) or 50 ng/ ⁇ l (E2) in an eppendorf tube.
  • the tubes containing plasmid DNA were centrifuged for 15 minutes at 1,400 rpm. the tubes were set on ice prior to microinjection. A 2 ⁇ L aliquot of plasmid DNA solution was loaded onto a femto top.
  • the tip was set with coverslip at 45°C, the pressure on the microinjector was set at 80 and the injection was performed.
  • the coverslips were incubated at 37°C overnight after injection. At 16 hours post-injection, cells were fixed and immunostained with goat anti-E7 polyclonal antibody, as explained below.
  • E2 or E7 Expression level of E2 or E7 was assessed using a fluorescent antibody assay as follows.
  • Coverslips were fixed in 10% formaldehyde in PBS for 20 minutes at room temperature and then washed twice with PBS.
  • goat anti-HPV-11 E7 or HPV-11 E2 serum was preabsorbed with VERO cells as follows. Confluent VERO cells from two T-150 flasks were scraped and then washed twice with PBS. then 200 ⁇ l serum was added to the cell pellet and mixed at 40°C overnight. the mixture was centrifuged; the supernate was removed to a new tube. the preabsorbed serum was stored in 50% glycerol at -20°C. Coverslips were incubated with goat anti-HPV-11 E7 or HPV-11 E2 protein serum preabsorbed as set forth below at a 1:1000 dilution in PBS for two hours at room temperature. Coverslips were washed with PBS three times, five minutes per wash.
  • Coverslips were incubated with FITC-conjugated Donkey Anti-Goat IgGAb (Jackson, ImmunoResearch, Cat #705-095-147) at 1:200 dilution in PBS. Coverslips were washed with PBS three times and then air-dried. Coverslips were mounted with 50% glycerol on slide glass. Coverslips were then examined under UV lights.
  • E2 RNA was prepared by transcribing plasmid pRc/CMV- 11E2 with T7 RNA polymerase using an Ambion MegaScript kit, following the manufacturer's directions.
  • E2 Translation of E2 was evaluated after separation of the translation mix by SDS-PAGE, followed by phospho-image analysis. To determine the effect of oligomers targeted to the translation initiation codon of E2, in vitro transcribed E2 mRNA was translated in the presence of 0.02 or 0.04 units/ul of RNAse H, and using
  • oligonucleotide concentrations ranging from 0.01 to 10 uM.
  • CAT mRNA was co-translated, or translated in independent translation reactions as control.
  • phosphodiester oligomers (compare the activity and specificity of oligomer 3102-1 [SEQ. ID. NO. 52] with 3233-1 [SEQ. ID. NO. 60] or 3234-1 [SEQ. ID. NO. 61]).
  • oligonucleotide backbone influenced the degree of antisense activity and specificity of the oligomer.
  • E1 mRNA was prepared by transcribing plasmid pRc/CMV-11E1 with T7 RNA polymerase using an Ambion MegaScript kit or an Ambion Message Machine kit, following the manufacturer's directions. In vitro transcribed E1 mRNA was cell-free
  • E1 mRNA was translated in the presence or absence of oligomers 2555-1 [SEQ. ID. NO. 40], 2556-1 [SEQ. ID. NO. 41], 2557-1 [SEQ. ID. NO. 42], 2744-1 [SEQ. ID. NO. 47], 3105-1 [SEQ. ID. NO. 43],
  • steric blocker oligomers 3105- 1 and 3196-1 showed low potency, and high specificity, while phosphodiester 2555-1, 2556-1, and 2557-1 produced high levels of inhibition of E1 translation at 1 uM, and different levels of non-specific translation inhibition of the CAT control.
  • oligomer 2557-1 targeted to the splice donor site at N847 showed less non-specific effects and oligonucleotide 2744-1, a 2'-OMe end-capped diester, was the most potent and specific.
  • the preferred target sequence around the translation initiation codon of E1 was AUG+14 and AUG+33 regarding oligomer backbones, a chimeric backbone is preferable.
  • E6/E7 mRNA 50nM was cell-free translated in rabbit reticulocyte lysates (Promega) as described in Example F. Cell-free translation was performed at 37°C for one hour and was stopped by addition of SDS gel loading buffer and incubation at 95°C for 3 minutes. Translation of E6 was evaluated after separation of the translation mix by SDS-PAGE analysis, followed by phospho-image analysis.
  • CaSki cells obtained from ATCC, Catalog CRL 1550 were seeded at 4 X 10 5 cells/60 mm dish and cultured in cell culture medium (90% RPMI, 10% fetal bovine serum, 50 I.U./mL penicillin and 50 mg/mL streptomycin). After 28 hours the cells were approximately 50% confluent.
  • RNAzolTMB TEL-TEST, Inc.
  • RNA probe used in the assay complements HPV-16 N566 to N855, so that if E7 mRNA is present, a protected band of 289 nucleotides should be expected.
  • the intracellular levels of actin mRNA were determined by probing an aliquot of the intracellular RNA with a commercially available probe targeted to actin (Ambion, pTRI- ⁇ -actin-125-Human).
  • oligomer 3678 [SEQ. ID. NO. 99] specifically inhibits HPV-16 E7 mRNA levels in transformed CaSki cells.
  • CaSki cells seeded in 60 mm dishes and cultured as described in Example R were transfected using 18 ug/mL of Lipofectamine (BRL) and 1 uM of oligomers 3678 [SEQ. ID. NO. 99], 3679 [SEQ. ID. NO. 100], 3680 [SEQ. ID. NO. 101] or control oligomer [SEQ. ID. NO. 102], as
  • cytoplasmic RNA was extracted as described before.
  • the intracellular levels of HPV-16 E7 mRNA after treatment with oligomers were determined using a RNase protection assay as described in Example R. As control for specificity, the intracellular levels of GAPDH mRNA were determined by probing an aliquot of the

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Abstract

Cette invention concerne des oligomères non codants qui sont complémentaires d'une région cible d'un ARNm ou d'un pré-ARN d'un virus du papillome humain et qui sont capables de s'hybrider sur cette région cible. Des régions cibles appropriées comprennent des séquences sélectionnées entre un codon d'initiation de transduction, un site donneur d'épissure, un site accepteur d'épissure, une région de codage, un signal de polyadénylation, une région 3' non traduite ou une région 5' non traduite d'un gène de virus du papillome humain.
PCT/US1995/005179 1994-04-26 1995-04-25 Oligomeres non codants utilises pour inhiber les virus du papillome humains WO1995028942A1 (fr)

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USPCT/US94/13387 1994-11-16
PCT/US1994/013387 WO1995013834A1 (fr) 1993-11-16 1994-11-16 Composes oligonucleosidiques chimeriques
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Cited By (8)

* Cited by examiner, † Cited by third party
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WO1996039501A3 (fr) * 1995-06-06 1997-02-06 Hoffmann La Roche Oligonucleotides specifiques contre le papillomavirus humain
EP0774518A2 (fr) * 1995-11-15 1997-05-21 Gen-Probe Incorporated Sondes d'acides nucléiques complémentaires aux acides nucléiques du virus du Papillome humain, procédés associés et trousse d'essais
US6458940B2 (en) * 1995-06-06 2002-10-01 Hybridon, Inc. HPV-specific oligonucleotides
DE10154831A1 (de) * 2001-11-08 2003-06-05 Deutsches Krebsforsch PNA-Konjugat oder PNA-Konjugat-Gemisch zur Therapie von mit HPV in Zusammenhang stehenden Erkrankungen
US7575918B2 (en) 1999-04-14 2009-08-18 The Penn State Research Foundation Tissue-specific and pathogen-specific ribozymes
US7704965B2 (en) 2002-06-26 2010-04-27 The Penn State Research Foundation Methods and materials for treating human papillomavirus infections
CN107723299A (zh) * 2011-10-12 2018-02-23 宾夕法尼亚大学理事会 用于人乳头状瘤病毒的疫苗及其使用方法
US20210355552A1 (en) * 2020-05-13 2021-11-18 Brigham Young University Paper-based colorimetric covid-19/sars-cov-2 test

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WO1991008313A1 (fr) * 1989-12-04 1991-06-13 Isis Pharmaceuticals, Inc. Inhibiteurs oligonucleotidiques non codants du virus du papillome
WO1993020095A1 (fr) * 1992-03-31 1993-10-14 Isis Pharmaceuticals, Inc. Inhibition du virus du papillome a l'aide d'oligonucleotides non codants

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WO1991008313A1 (fr) * 1989-12-04 1991-06-13 Isis Pharmaceuticals, Inc. Inhibiteurs oligonucleotidiques non codants du virus du papillome
WO1993020095A1 (fr) * 1992-03-31 1993-10-14 Isis Pharmaceuticals, Inc. Inhibition du virus du papillome a l'aide d'oligonucleotides non codants

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ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Vol. 37, No. 2, issued Feb. 1993, COWSERT et al., "In Vitro Evaluation of Phosphorothioate Oligonucleotides Targeted to the E2 mRNA of Papillomavirus: Potential Treatment for Genital Warts", pages 171-177. *
CLINICAL RESEARCH, Vol. 39, No. 4, issued 1991, COWSERT et al., "Antisense Oligonucleotides as Inhibitors of Papillomavirus", page 818A. *
INT. J. CANCER, Vol. 51. No. 5, issued 09 July 1992, VON KNEBEL BOEBERITZ et al., "Inhibition of Tumorigenicity of Cervical Cancer Cells in Nude Mice by HPV E6-E7 Anti-Sense RNA", pages 831-834. *
NUCLEIC ACIDS RESEARCH, Vol. 19, No. 15, issued 1991, STOREY et al., "Anti-Sense Phosphorothioate Oligonucleotides Have Both Specific and Non-Specific Effects on Cells Containing Human Papillomavirus Type 16", pages 4109-4114. *

Cited By (15)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6509149B2 (en) 1995-06-06 2003-01-21 Hybridon, Inc. HPV-specific oligonucleotides
WO1996039501A3 (fr) * 1995-06-06 1997-02-06 Hoffmann La Roche Oligonucleotides specifiques contre le papillomavirus humain
US6458940B2 (en) * 1995-06-06 2002-10-01 Hybridon, Inc. HPV-specific oligonucleotides
US7470512B2 (en) 1995-11-15 2008-12-30 Gen-Probe Incorporated Oligonucleotides for use in determining the presence of human papilloma virus in a test sample
US7355034B2 (en) 1995-11-15 2008-04-08 Gen-Probe Incorporated Oligonucleotides for use in determining the presence of human papilloma virus in a test sample
EP0774518A2 (fr) * 1995-11-15 1997-05-21 Gen-Probe Incorporated Sondes d'acides nucléiques complémentaires aux acides nucléiques du virus du Papillome humain, procédés associés et trousse d'essais
US7875441B2 (en) 1995-11-15 2011-01-25 Gen-Probe Incorporated Oligonucleotides for detecting human papilloma virus in a test sample
US8501410B2 (en) 1995-11-15 2013-08-06 Gen-Probe Incorporated Oligonucleotides for detecting human papilloma virus in a test sample
US9194008B2 (en) 1995-11-15 2015-11-24 Gen-Probe Incorporated Hybridization assay detection probes for detecting human papilloma virus in a sample
US7575918B2 (en) 1999-04-14 2009-08-18 The Penn State Research Foundation Tissue-specific and pathogen-specific ribozymes
DE10154831A1 (de) * 2001-11-08 2003-06-05 Deutsches Krebsforsch PNA-Konjugat oder PNA-Konjugat-Gemisch zur Therapie von mit HPV in Zusammenhang stehenden Erkrankungen
US7704965B2 (en) 2002-06-26 2010-04-27 The Penn State Research Foundation Methods and materials for treating human papillomavirus infections
CN107723299A (zh) * 2011-10-12 2018-02-23 宾夕法尼亚大学理事会 用于人乳头状瘤病毒的疫苗及其使用方法
CN107723299B (zh) * 2011-10-12 2022-02-01 宾夕法尼亚大学理事会 用于人乳头状瘤病毒的疫苗及其使用方法
US20210355552A1 (en) * 2020-05-13 2021-11-18 Brigham Young University Paper-based colorimetric covid-19/sars-cov-2 test

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