WO1996029337A1 - Phosphorothioates d'oligodesoxynucleotides anti-sens modifies de thiono-triester - Google Patents

Phosphorothioates d'oligodesoxynucleotides anti-sens modifies de thiono-triester Download PDF

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Publication number
WO1996029337A1
WO1996029337A1 PCT/US1996/003843 US9603843W WO9629337A1 WO 1996029337 A1 WO1996029337 A1 WO 1996029337A1 US 9603843 W US9603843 W US 9603843W WO 9629337 A1 WO9629337 A1 WO 9629337A1
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oligonucleotide
nucleic acid
infectious agent
expression
gene
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PCT/US1996/003843
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English (en)
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Zhaoda Zhang
Jimmy X. Tang
Jin Yan Tang
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Hybridon, Inc.
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Priority to JP8528609A priority Critical patent/JPH11502818A/ja
Priority to AU53193/96A priority patent/AU5319396A/en
Publication of WO1996029337A1 publication Critical patent/WO1996029337A1/fr

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    • 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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/06—Antihyperlipidemics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals

Definitions

  • This invention relates to the field of modified oligodeoxynucleotides, methods for their synthesis, and methods of their use to inhibit gene expression.
  • Antisense oligonucleotides and their modified analogs have been shown to regulate the expression of genes (Zamecnik in Prospects for Antisense Nucleic Acid Therapy for Cancer and AIDS, pp. 1-6 (Wickstrom, E, Ed , Wiley Liss, N Y., 1991); Agrawal, Trends in Biotechnology 10, 152-158 (1992); Wickstom, Trends in Biotechnology 10, 281-286 (1992); Rapaport et al., Proc. Natl. Acad Sci. USA 89, 8577-8580 (1992); and Agrawal, S. (1991) in Prospects for Antisense Nucleic Acid Therapy for Cancer and AIDS, pp.
  • Enzymatic degradation of oligonucleotide phosphorothioates is primarily from the 3' end
  • oligonucleotides exhibit strong cellular uptake, high stability when bound to the target, and high resistance to nuclease attack.
  • the present invention comprises a novel class of modified antisense oligonucleotides having increased cellular uptake, high stability when bound to the target nucleic acid, and high resistance to nuclease attack.
  • novel antisense oligonucleotides are characterized by having one to ten thiono triester phosphorothioate intemucleotide linkages. These intemucleotide linkages have the following structure:
  • R is a large lipophilic moiety, such as a straight or branched chain alkyl group, a cholesteryl derivative, or an adamantyl derivative.
  • Oligonucleotides having one or more of these types of linkages exhibit increased resistance to degradation by T4 polymerase and DNA polymerase I. They also exhibit increased melting temperatures when hybridized to target nucleic acids as compared to oligonucleotide phosphorothioates.
  • the oligonucleotides of the present invention are useful for both in vitro and in vivo suppression of nucleic acid expression.
  • in vitro uses for the present oligonucleotides is the determination of the role particular proteins play in biological processes by modulating the expression of the genes encoding the protein under study.
  • the elucidation of most biochemical pathways now known was accomplished by isolating and studying deletion mutants in vitro Studying deletion mutants is arduous, however.
  • the presently claimed oligonucleotides provide an attractive alternative because it is much less laborious to modulate gene expression using antisense oligonucleotides than it is using the deletion mutation approach.
  • the oligonucleotides of the present invention are useful research tools.
  • the oligonucleotides of the present invention are suitable for in vivo use as well.
  • the increased stability of the oligonucleotide-target hybridization complex, the increased resistance to nuclease attack, and the increased susceptibility to cellular uptake all make the oligonucleotides -4- of the present invention ideal for treating infections by a wide variety of diseases caused by pathogens, including viral and bacterial agents.
  • the presently disclosed oligonucleotides will have at least one region whose sequence is sufficiently complementary to a region of the pathogen's nucleic acid (the target nucleic acid) to result in hybridization to the target nucleic acid and suppression of its expression, both under intracellular conditions.
  • the presently claimed oligonucleotides are also useful for treating diseases arising from genetic abnormalities that cause under- or over- expression of a gene.
  • the presently claimed oligonucleotides may be designed to target the abnormal gene directly, or, in the alternative, to target the gene encoding the protein that promotes expression of the abnormal gene
  • the presently claimed oligonucleotides are useful both in vitro and in vivo in essentially any situation in which one desires to modulate gene expression
  • the present invention also provides novel compositions comprising the inventive oligonucleotides as well as methods for employing the oligonucleotides to treat pathogenic diseases and other abnormal states arising from aberrant gene expression
  • inventive oligonucleotides as well as methods for employing the oligonucleotides to treat pathogenic diseases and other abnormal states arising from aberrant gene expression
  • Figure 1 displays the synthetic scheme for producing phosphoramidite intermediates useful in the production of oligonucleotides bearing S-triester intemucleotide linkages.
  • Figure 2 displays an autoradiogram of the digestion of modified S-triester- phosphorothioate oligonucleotides by T4 polymerase.
  • Figure 3 displays an autoradiogram of the digestion of an O-ethyl triester phosphorothioate by DNA polymerase I.
  • Figure 4 displays an autoradiogram of the digestion of an ordinary phosphorothioate and an O-ethyl triester phosphorothioate oligonucleotide by T4 polymerase.
  • the present invention comprises, inter alia, a new class of antisense oligonucleotides having improved properties relative to prior art antisense oligonucleotides.
  • the present oligonucleotides exhibit increased susceptibility to cellular uptake when carrying a large lipophilic group, increased resistance to exonucleolytic degradation, and increased stability of hybridization complexes formed between the oligonucleotides and their targets. These properties make these novel oligonucleotides ideal for modulating gene expression, both in vitro and in vivo.
  • Oligonucleotides according to the invention may be anywhere from 2 to 100 nucleotides in length. In a preferred embodiment, the oligonucleotides will be from 13 to 50 nucleotides in length. In a more preferred embodiment, the oligonucleotides will be from 20 to 35 nucleotides in length.
  • R is any large lipophilic group that doesn't substantially change the geometry of the intemucleotide bond in a manner that dimishes the oligonucleotide 's efficacy at modulating gene expression.
  • R is a C,-C 22 linear or branched chain alkyl group, a cholesteryl derivative having the structure:
  • n 2-12, a 1,2-di-O- alkyl-rac-3-glyceryl derivative having the structure:
  • the R groups of different linkages may be the same or different.
  • R is chosen from the group consisting of l-adamantyl-2- ethyl, 1-hexadecyl, and cholesteryl-3-carboxyamino-6-hexyl.
  • Oligonucleotides according to the invention are synthesized via a precursor having the following structure: -7-
  • R is as described above
  • B is a suitably protected (if necessary) base
  • Figure 1 displays the scheme for synthesizing these intermediates and representative protocols are provided in the Examples, infra.
  • the oligonucleotide will have from 1 to 10 S-triester phosphorothioate intemucleotide linkages.
  • the oligonucleotides will have an S-triester phosphorothioate intemucleotide linkage at the 3' or 5' terminal linkage, or at both the 3 ' and 5' terminal linkages.
  • all of the S-triester linkages may have the same R substituent or they may all be different.
  • the oligonucleotides of the present invention exhibit increased nuclease resistance and, when having a bulky lipophilic group, exhibit substantially increased cellular uptake relative to the corresponding phosphorothioate intemucleotide analogs while still maintaining the ability to specifically hybridize to a complementary target nucleic acid under normal stringency conditions.
  • Increases in melting temperature (T,.) of 5 °C and more have been observed with a corresponding increase in cellular uptake of 2.5 to 3 times that of the phosphorothioate analog.
  • the antisense oligonucleotides of the present invention may be designed to incorporate a number of additional features that have been demonstrated to increase efficacy. For example, they may be designed to be "self-stabiliaZed," i.e., having a first region sufficiently complementary to a second region to allow for intramolecular hybridization, thereby rendering the oligonucleotide less susceptible to nucleolytic attack.
  • self-stabiliaZed i.e., having a first region sufficiently complementary to a second region to allow for intramolecular hybridization, thereby rendering the oligonucleotide less susceptible to nucleolytic attack.
  • the presently disclosed oligonucleotides may be designed to be "fold-back triplex forming," i.e., having a first region complementary to a target nucleic acid and a second region having a sequence that allows for triplex formation by Hoogsteen base pairing between it and the duplex formed by the first region and the target nucleic acid, as described in PCT Intemational Application Publication No. WO 94/17091.
  • Oligonucleotides according to the invention are useful for both in vitro and in vivo applications.
  • the present oligonucleotides are useful as research tools in determining gene function. Because they can be prepared to be complementary to a particular sequence, the present oligonucleotides can be used to selectively inhibit expression of a target gene.
  • the present oligonucleotides thus provide an attractive and easily used alternative to the laborious method of gene inhibition by mutation (e.g., deletion mutation). The significance of this will be appreciated when one realizes that the elucidation of all biological pathways now known was determined by deletion mutations.
  • the oligonucleotides of the present invention are also useful as therapeutic agents for diseases or physiological conditions involving expression of specific genes.
  • Oligonucleotides useful for treating a disease or condition will have a nucleotide sequence sufficiently complementary to the target nucleic acid to bind under physiological conditions.
  • the terms "complementary” and “sufficiently complementary” are used interchangably and, when used to describe the sequence of an antisense oligonucleotide, mean that the oligonucleotide sequence is such that the oligonucleotide inhibits expression of the target nucleic acid under the conditions of interest (e.g., in vitro experimental conditions and physiological conditions).
  • oligonucleotides according to the invention will have sequence complementary to a nucleic add (e.g., a gene or mRNA) that is essential to a biological process.
  • oligonucleotides of the invention can also be complementary to a gene or other nucleic acid whose expression causes or is involved in a diseased or otherwise abnormal state of the organism. Because of their efficacy at gene modulation, the presently claimed oligonucleotides are also useful for treating diseases arising from genetic abnormalities that cause under- or over- expression of a gene.
  • the presently claimed oligonucleotides may be designed to target the abnormal gene directly, or, in the alternative, to target the gene encoding the protein that promotes expression of the abnormal gene. Conversely, where an abnormal gene is under-expressed, one may design an oligonucleotide that suppresses expression of a gene encoding a protein that suppresses expression of the abnormal gene.
  • the target nucleic acid sequence will be a virus nucleic acid sequence.
  • the use of antisense oligonucleotides to inhibit various viruses is well known and has been reviewed in Agrawal, Tibtech 10, 152 (1992).
  • Viral nucleic acid sequences that hybridize to effective antisense oligonucleotides have been described for many viruses, including human immunodeficiency vims type 1 (U.S. Patent No. 4,806,463), Herpes simplex virus (U.S. patent No. 4,689,320), Influenza virus (U.S. Patent No. 5,194,428), and Human papilloma virus (Storey et al., Nucleic Acids Res. 19:4109-4114 (1991)).
  • nucleotide sequences complementary to nucleic acid sequences from any other virus can be used, as can nucleotide sequences complementary to nucleic acid sequences from any other virus.
  • Additional viruses that have known nucleic acid sequences against which an antisense oligonucleotide according to the invention can be prepared include, but are not limited to, Foot and Mouth Disease Virus (See Robertson et al., J. Virology 54, 651 (1985); Harris el al., J. Virology 36, 659 (1980)), Yellow Fever Virus (See Rice et al., Science 229, 726 (1985)), Varicella-Zoster Vims (See Davison and Scott, J. Gen.
  • the oligonucleotides of the invention can have a nucleotide sequence complementary to a nucleic acid sequence of a pathogenic organism.
  • the nucleic acid sequences of many pathogenic organisms have been described, including the malaria organism, Plasmodium falciparum, and many pathogenic bacteria.
  • pathogenic eukaryotes having known nucleic acid sequences against which oligonucleotides of the present can be prepared include, but are not limited to Trypanosoma brucei gambiense and Leishmania (See Campbell et al., Nature 311, 350 (1984)), and Fasciola hepatica (See Zurita et al., Proc. Natl. Acad. Sci. USA 84, 2340 (1987)).
  • Antifungal oligonucleotides can be prepared having a nucleotide sequence that is complementary to a nucleic acid sequence from, e.g., the chitin synthetase gene, and antibacterial oligonucleotides according to the invention can be prepared using, e.g., the alanine racemase gene.
  • the oligonucleotides can have a nucleotide sequence complementary to a cellular gene or gene transcript, the abnormal expression or product of which results in a disease state.
  • the nucleic acid sequences of several such cellular genes have been described, including prion protein (Stahl and Prusiner, FASEB J. 5, 2799 ( 1991 )), the amyloid-like protein associated with Alzheimer's disease (U.S. Patent No. 5,015,570), and various well-known oncogenes and proto-oncogenes, such as c-myb, c-myc, c-abl, and n-ras.
  • oligonucleotides that inhibit the synthesis of structural proteins or ertazymes involved largely or exclusively in spermatogenesis, sperm motility, the binding of the sperm to the egg or any other step affecting sperm viability may be used as contraceptives for men.
  • contraceptives for women may be oligonucleotides that inhibit production of proteins or enzymes involved in ovulation, fertilization, implantation or in the biosynthesis of hormones involved in those processes.
  • Hypertension can be controlled by oligonucleotides that suppress the synthesis of angiotensin converting enzyme or related enzymes in the renin/angiotensin system; platelet aggregation can be controlled by suppression of the synthesis of enzymes necessary for the synthesis of thromboxane A2 for use in myocardial and cerebral circulatory disorders, infarcts, arteriosclerosis, embolism and thrombosis; deposition of cholesterol in arterial wall can be inhibited by suppression of the synthesis of fatty acyl co-enzyme A: cholesterol acyl transferase in arteriosclerosis; inhibition of the synthesis of cholinephosphotransferase may be useful in hypolipidemia.
  • oligonucleotides of the present invention can be used to reduce or eliminate adverse effects of the disorder.
  • suppression of the synthesis of monoamine oxidase can be used in Parkinson's disease
  • suppression of catechol O-methyl transferase can be used to treat depression
  • suppression of indole N-methyl transferase can be used in treating schizophrenia.
  • Suppression of selected enzymes in the arachidonic acid cascade may be useful in the control of platelet aggregation, allergy, inflammation, pain and asthma.
  • nucleotide sequences complementary to nucleic acid sequences from any of these genes can be used for the oligonucleotides according to the invention, as can be oligonucleotide sequences complementary to any other cellular gene or gene transcript, the abnormal expression or product of which results in a disease state.
  • a variety of viral diseases may be treated by oligonucleotides having one or more S-triester phosphorothioates intemucleotide linkages, including aAIDS, aARC, oral or genital herpes, papilloma warts, flu, foot and mouth disease, yellow fever, chicken pox, shingles, HTLV- leukemia, and hepatitis.
  • oligonucleotides Among fungal diseases treatable by oligonucleotides according to the invention are candidiasis, histoplasmosis, cryptococcocis, blastomycosis, aspergillosis, sporotrichosis, chromomycosis, dematophytosis and coccidioidomycosis.
  • the method can also be used to treat rickettsial diseases (e.g., typhus, Rocky Mountain spotted fever), as well as sexually transmitted diseases caused by Chlamydia trachomatis or Lymphogranuloma venereum.
  • a variety of parasitic diseases can be treated by oligonucleotides of the present invention, including amebiasis, Chegas' disease, toxoplasmosis, pneumocystosis, giardiasis, cryptosporidiosis, trichomoniasis, and Pneumocystis carini pneumonia; also worm (helminthic diseases) such as ascariasis, filariasis, trichinosis, schistosomiasis and nematode or cestode infections. Malaria can be treated by oligonucleotides of the present invention, regardless of whether h is caused by P.f ⁇ lcip ⁇ rum, P. viv ⁇ x, P. or ⁇ le, or P.
  • oligonucleotides according to the invention can be prepared having a nucleotide sequence that hybridizes to a nucleic acid sequence that is an essential nucleic acid sequence for the propagation of the infectious agent, such as an essential gene.
  • an essential gene or nucleic acid is one that is required for a biological process and without which the biological process does not occur.
  • Anhydrous acetonitrile, tetrahydrofuran, dichloromethane, ethyl alcohol, 2-propanol, pentane, triethylamine, 1-hexadecanol, tetrazole, cholesteryl chloroformate, 6-arnino-l-hexanol, bis(diisopropylamino)chlorophosphine and 1 -adamantaneethanol were purchased from Aldrich (Milwaukee, WI). Hexane, ethyl acetate, and methanol were purchased from J.T. Baker Inc.
  • Fluorescein-ON phosphoramidite was purchased from CLONTECH Laboratories, Inc. (Palo Alto, CA).
  • the reagents (Sequagel Sequencing System: concentrate, diluent and buffer) used for PACE were purchased from National Diagnostics (Atlanta, GA).
  • 31 P NMR spectra 121.65 MHz were recorded on a Varian (Palo Alto, CA) UNITY 300 (the chemical shift was correlated to 85% H 3 PO 4 ).
  • Thermal melting data were collected from GBC 920 UV-Vis spectrophotometer (Dandenong, Victoria 3175, Australia).
  • Oligonucleotide synthesis was performed on a 8909 Expedite DNA synthesizer (Mllipore). PAGE was carried out by using Model S2 sequencing gel electrophoresis apparatus (LLFE TECHNOLOGY, Gaithersburg, MD) and EC600-90 power supply (E-C APPARATUS COPORATION, St. Russia, FL). Fluorescence was measured by Spectrofluorometer (PTI Technology, South Brunswick, N.J ). The UV absorptance was measured UV-160A UV spectrometer (SHIMADZU, Columbia, MD).
  • O-(cholesteryl-3-carboxy-amino-6-hexyl)- phosphordiamidite was obtained as a colorless sticky oil (4 38 g, 98 4 % yield) by using bis(diisopropylam ⁇ no)chlorophosphine ( 1 56 g, 5 84 mmol), triethylamine (1.22 ml, 0 89 g, 8 76 mmol), cholesteryl-3-carboxyamino-6-hexanol (3 10 g, 5 84 mmol) and THF (3 6 ml), 31 P NMR (CDC1 3 ) ⁇ 136 17
  • O-ethyl-phosphordiamidite (1.02 g, 3.67 mmol) was added in one batch under nitrogen to a stirred suspension of 5'-DMT-T (1.0 g, 1. 84 mmol) in CH 2 C1 2 (5.0 ml)
  • a solution of tetrazole (0.129 g, 1.84 mmol) in acetonitrile (4.7 ml) was added dropwise to the resulting mixture. After the mixture was stirred for 5 hours at room temperature, the solvent was removed at reduced pressure to give a foamy solid.
  • the cmde product was purified by flash column chromatography (eluant: CH ⁇ l j /EtOAc/E ⁇ N 70:20: 10) and precipitated from hexane at -78 °C to give 5'-DMT-dT-O- ethyl-phosphoramidite as a white foam (0.62 g, 47.0% yield): TLC R 7 0.66 (CH 2 Cl 2 EtOAc NEt 3 45:45: 10); 31 P NMR (CDC1 3 ) ⁇ 157.73, 158.52.
  • O-ethyl-phosphordiamidite (1.71 g, 4.64 mmol) in CH 2 C1 2 (9.7 ml) was added under nitrogen to a stirred pale yellow suspension of 5'-DMT-dC' BA (2.25 g, 3.08 mmol) in CH 2 C1 2 (11.3 ml).
  • a solution of tetrazole (0.216 g, 3.08 mmol) in acetonitrile (9.7 ml) was added dropwise to the resulting mixture. After the mixture was stirred for 5 hours at room temperature, the solvent was removed at reduced pressure to give a foamy solid.
  • O-isopropyl-phosphordiamidite (0.80 g, 2.8 mmol) in CH 2 C1 2 (2.0 ml) was added under nitrogen to a stirred suspension of 5'-DMT-T (1.0 g, 1.9 mmol) in CH 2 C1 2 (5.0 ml).
  • a solution of tetrazole (0.129 g, 1.84 mmol) in acetonitrile (5.7 ml) was added to the resulting mixture.
  • the cmde product was purified by flash column chromatography (eluant: CHjClz/EtOAc/Et 70:20: 10) to give 5'-DMT-dT-O(cholesteryl-3-carboxyamino-6-hexyl)-phosphoramidite as a white foam (2.11 g, 95.2% yield): TLC R 7 0.60 (CH ⁇ tOAc/NE ⁇ 45:45:10); 3, P NMR (CDC 1 3 ) ⁇ 158.60, 158.82.
  • the cmde product was purifi-sd by fl-ash column chromatography (eluant: CH 2 Cl 2 /Et ⁇ Ac/Et 3 N 70:20: 10) to give 5'-DMT-dC fflA -O-(l-hexadecyl)-phosphoramidite as a pale yellow sticky oil (1.41 g, 93.5% yield): TLC R f 0.62 (CH 2 Cl 2 Et ⁇ Ac/NEt 3 45:45: 10), 31 P NMR (CDC1 3 ) ⁇ 158.71, 159.47.
  • O-alkyl-phosphoramidite The syntheses of a number of O-alkyl-phosphoramidites have been reported, which include methyl, ethyl, trifluoroethyl, isopropyl and neopentyl derivatives. Koziolkiewicz and Wilk in Protocols for Oligonucleotides and Analogs, supra, pp. 207-224, and references cited therein In a similar way, the O-alkyl-phosphordiamidites were prepared by the reaction of bis(diisopropylamino)chlorophosphine with the alcohols.
  • the S-triester-phosphorothioate and O-triester-phosphodiester oligonucleotides were synthesized using the phosphoramidites of Example 17 protected by the base labile tert- butylphenoxyacetyl (tBA) group on the exocyclic amine (dA, dC, and dG).
  • tBA base labile tert- butylphenoxyacetyl
  • Oligonucleotides were purified by reverse-phase HPLC and/or PAGE.
  • the 31 P NMR spectmm of the S-triester-phosphorothioate compound no. 1.1 showed that the corresponding peaks for S-triester-phosphorothioate and S-phosphorothioate intemucleotide linkages appear at 62 and 53 ppm respectively.
  • the ratio between S-triester and phosphorothioate was 19.8 : 100.0 (The calculated value is 20:100).
  • the content of O- phosphodiester linkages was shown to be less than 2.5 percent.
  • 5'-DMT-dT-O-iso-propyl-phosphoramidite, and 5'-DMT-dC lBA -O-isopropyl-phosphoramidite were dissolved in dry acetonitrile at a concentration of 50 mg ml, and the others, 5'-DMT-dT-O-( l -adamantyl-2-ethyl)- phosphoramidite, 5'-DMT-dC ,BA - ad amantyl-2 -ethyl )- p ho sp ho rami dit e , 5 ' - DMT- dT - O- (chol e st eryl -
  • 3-carboxyamino-6-hexyl)-phosphoramtidite, 5'-DMT-dC lB ⁇ -O-(cholesteryl-3-carboxyamino-6- hexyl)-phosphoramidite, 5'-DMT-dT-O-(l-hexadecyl)-phosphoramidite, and 5VDMT- dC lBA -O-(l-hexadecyl)-phosphoramidite were dissolved in dichloromethane and diluted with acetonitrile to acetonitrile-dichloromethane (1 :1) at a final concentration of 50 mg/ml.
  • oligonucleotides For phosphorothioate oligonucleotides, the iodine oxidation step was replaced by sulfurization with 3H-l,2-benzodithiol-3-one-l,l-dioxide (Beaucage reagent). Iyer et al., J. Org. Chem 55, 4693 (1990). Two-hour treatment with ammonium hydroxide at room temperature was carried out to cleave the oligomer from the support .and to deprotect nucleoside bases Oligonucleotides were purified by reverse-phase HPLC and/or PAGE, and desalted by using C- 18 SEP-PAK cartridges
  • Fluorescein labeling of oligonucleotides Fluorescein was conjugated to the 5' end of the oligonucleotides by either an automated DNA synthesizer or by a manual procedure using a FLUORESCEIN-ONTM phosphor.amidite The efficiency of fluorescein labeling was determined by using a spectrofluorometer (excitation 488 nm, emission 520 nm)
  • Example 20 Exonuclease resistance of triester containing oligonucleotides Sensitivity to 3 -exonuclease degradation was measured by digestion with T4 DNA polymerase and/or DNA polymerase I The oligonucleotides tested are listed in Table 1
  • oligonucleotides (30 pmole) were dissolved in 20 ⁇ l of buffer (50 mM Tris, pH 8.0, MgCl 2 , 5 mM DTT, 0.05% BSA) and incubated with DNA polymerase I (5.0 units) at 37 °C Aliquots (5 ⁇ l), inhibited by 6 ⁇ l of the stop solution (95% foimamide, 10 mM EDTA, 0.05% bromophenol blue, 0.05% xylene cyanol), were removed at 0, 60, 120, 180 and 240 minutes, analyzed by PAGE (20% polyacrylamide containing 8.3 M urea), and followed by autoradiography. Typical results are shown in Fig. 3.
  • TJ Melting Temperatures were determined for the duplexes of unmodified and modified oligonucleotides with the complementary DNA.
  • Each oligonucleotide (0.2 A 260 Units) and its complementary DNA was annealed in 1 ml buffer (10 mM Na 2 HPO 4 , pH 7.4, 0.1 M NaCl) by heating to 80 °C and then cooling down to 40 °C at a rate of 2 °C/minute. The mixture was then reheated to 80 °C at a rate of 1 ° C/minute and the A 260 was continuously recorded. Melting profiles were obtained for oligonucleotides.
  • S-phosphorothioate reduces the total negative charge of the oligonucleotide, which would be expected to enhance hybridization.
  • Table 2 shows, almost all of the modified S- triester-phosphorothioates have increased T m compared to the unmodified phosphorothioate
  • Example 22 Cellular Uptake Cell culture Human T cell and leukemia cell line H9 were used in the study. They were cultured in RPMI media supplemented with 10% fetal bovine semm (heat inactivated to 56 °C for 30 minutes to inactivate the nucleases), 2 mM glutamine, 100 ⁇ l streptomycin, 100 U/mL penicillin and 6 x 10 '5 M of 2-mercaptoethanol in an air incubator (37 C, humidified by 5% CO 2 -95% O 2 )
  • Oligonucleotide complexes (0 2 OD/100 ⁇ l) were added to the cells (5 x 10 6 cells/ml, 0 5 ml) and set to culture aAfter 4 hours of culture, aliquots of cell culture mixtures were removed, washed, and resuspended in Hank's balanced salt solution (HBSS) supplemented with 0 1 % BSA and 0 1 % sodium azide Propidium iodide (final concentration 10 ⁇ l/ml) was used to distinguish viable cells from dead cells.
  • HBSS Hank's balanced salt solution
  • All compounds are ohgodeoxyribonucleotides and are presented in the 5' to 3' direction, left to right; highlighted nucleotides have an S-triester linkage at the 3' side.
  • R 4 Cholesteryl-3-carboxyamino-6-hexyl
  • R 3 l-hexadecyl.
  • 5'-TCTGGGTAATTACACGCAAGC-3' an unrelated nonspecific control oligonucleotide
  • 5'-ACAAGCGTTCCATTCGGGGT-3' a nonsense control oligonucleotide that is the same nucleotide sequence as UL36 ANTI, except in reverse order.
  • HFF Human foreskin fibroblasts
  • the cells were incubated with HCMV strain AD 169 (ATCC VR-538) at a multiplicity of infection (MOI) of 0.1 for 1 hr at 37°C. Cells were washed again and refed in fresh growth medium containing serial dilutions of the antisense oligonucleotide at the same concentrations used in the preincubation. At 5 to 6 days postinfection (p i ), cells were fixed (100% ethanol) and reacted with a primary antibody specific for the HCMV UL44 gene product (Advanced Biotechnologies, Rivers Park, IL).
  • MOI multiplicity of infection
  • the cells were then reacted with anti-mouse immunoglobulin G conjugated to horseradish peroxidase-labeled (kerkgaard and Perry, Gaithersburg, MD) secondary antibody and developed with a TMB substrate, and the optical density at 450 nm was determined by using a plate reader (Ceres 900, Biotek, Winooski, VT). These ELISA experiments showed that some of these modified oligonucleotides almost completely inhibit HCMV DNA replication when used at concentrations as low as 0.08 ⁇ M. The results are presented in Table 5.
  • R group corresponds to one S-tnester or O-tnester linkage, with the first listed R group being in the 5 '-most S- tnester or O-tnester linkage and so on so that the last R group listed corresponds to the 3 -most S-tnester or O- tnester linkage

Abstract

Cette invention concerne des oligonucléotides anti-sens possédant des qualités d'absorption cellulaire accrues, une meilleure résistance à la nucléase, et des qualités de liaison à une cible plus stables sur le plan thermodynamique. Ces nouveaux oligonucléotides se caractérisent par le fait qu'ils possèdent de 1 à 10 liaisons d'internucléosides de phosphorothioate de thiono-triester ayant des moitiés lipophiles.
PCT/US1996/003843 1995-03-23 1996-03-22 Phosphorothioates d'oligodesoxynucleotides anti-sens modifies de thiono-triester WO1996029337A1 (fr)

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WO1998007734A1 (fr) * 1996-08-21 1998-02-26 Hybridon, Inc. Promedicaments oligonucleotidiques
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Publication number Priority date Publication date Assignee Title
WO1997033992A1 (fr) * 1996-03-14 1997-09-18 Hybridon, Inc. Oligonucleotides selectionnes avec activite anti-cytomegalovirus
US6776986B1 (en) 1996-06-06 2004-08-17 Novartis Ag Inhibition of HIV-1 replication by antisense RNA expression
WO1998004575A3 (fr) * 1996-07-31 1998-03-19 Gilead Sciences Inc Analogues lipophiles d'oligonucleotides
WO1998007734A1 (fr) * 1996-08-21 1998-02-26 Hybridon, Inc. Promedicaments oligonucleotidiques
US9358300B2 (en) 1998-11-12 2016-06-07 Life Technologies Corporation Transfection reagents
WO2006065751A2 (fr) * 2004-12-13 2006-06-22 Government Of The United States Of America, Represented By The Secretary, Department Of Health And Human Services Promedicaments d'oligonucleotides cpg, compositions afferentes et procedes therapeutiques associes
WO2006065751A3 (fr) * 2004-12-13 2006-12-07 Us Gov Health & Human Serv Promedicaments d'oligonucleotides cpg, compositions afferentes et procedes therapeutiques associes
US9809824B2 (en) 2004-12-13 2017-11-07 The United States Of America, Represented By The Secretary, Department Of Health And Human Services CpG oligonucleotide prodrugs, compositions thereof and associated therapeutic methods
US10195280B2 (en) 2014-07-15 2019-02-05 Life Technologies Corporation Compositions and methods for efficient delivery of molecules to cells
US10792362B2 (en) 2014-07-15 2020-10-06 Life Technologies Corporation Compositions and methods for efficient delivery of molecules to cells
US11872285B2 (en) 2014-07-15 2024-01-16 Life Technologies Corporation Compositions and methods for efficient delivery of molecules to cells

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