Antiviral oligonucleotides having a conserved G4 core sequence

Inactive Publication Date: 2007-01-18
IONIS PHARMA INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Benefits of technology

[0033] It has been discovered that oligonucleotides containing the sequence GGGG (G4), denominated herein as a conserved G4 core sequence, have antiviral activity against a number of viruses including but not limited to HIV, HSV, HCMV, and influenza virus. A sequence containing 4 guanines (G's) or 2 stretches of 3 G's has been found to be effective for significant antiviral activity. It has also been discovered that oligonucleotides containing a conserve

Problems solved by technology

Although each of the above publications have reported some degree of success in inhibiting some function of the virus, a general therapeutic scheme to target HIV and other viruses has not been found.
IDU is a moderately effective topical antiviral agent when applied to HSV gingivostomatitis and ocular stromal keratitis; however, its use in controlled clinical studies of HSV encephalitis revealed a high toxicity associated with IDU treatment.
Reports of clinical efficacy are contradictory and a major disadvantage for practical use is the extremely poor solubility of Ara-A in water.
However there appear to be both the existence of drug-resistant viral mutants and negative results in double-blind studies of HSV-1 treatment with ACV.
ACV, like Ara-A, is poorly soluble in water (0.2%) and this physical characteristic limits the application forms for ACV.
The practical application of purine nucleoside analogs in an extended clinical situation suffers from their inherently efficient catabolism, which not only lowers the biological activity of the drug but also may result in the formation of toxic catabolites.
The efficacy of these compounds is diminished by their inherently poor solubility in

Method used

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  • Antiviral oligonucleotides having a conserved G4 core sequence
  • Antiviral oligonucleotides having a conserved G4 core sequence
  • Antiviral oligonucleotides having a conserved G4 core sequence

Examples

Experimental program
Comparison scheme
Effect test

example 1

Oligonucleotide Synthesis

[0077] DNA synthesizer reagents, controlled-pore glass (CPG)-bound and B-cyanoethyldiisopropylphosphoramidites were purchased from Applied Biosystems (Foster City, Calif.). 2′-O-Methyl B-cyanoethyldiisopropylphosphoramidites were purchased from Chemgenes (Needham, Mass.). Phenoxyacetyl-protected phosphoramadites for RNA synthesis were purchased from BioGenex (Hayward, Calif.).

[0078] Oligonucleotides were synthesized on an automated DNA synthesizer (Applied Biosystems model 380B). 2′-O-Methyl oligonucleotides were synthesized using the standard cycle for unmodified oligonucleotides, except the wait step after pulse delivery of tetrazole and base was increased to 360 seconds. The 3′ base bound to the CPG used to start the synthesis was a 2′-deoxyribonucleotide. After cleavage from the CPG column and deblocking in concentrated ammonium hydroxide at 55° C. (18 hours), the oligonucleotides were purified by precipitation two times out of 0.5 M NaCl solution with...

example 2

HIV Inhibition Acute HIV Infection Assay

[0079] The human T-lymphoblastoid CEM cell line was maintained in exponential growth phase in RPMI 1640 with 10% fetal calf serum, glutamine, and antibiotics. On the day of the assay, the cells were washed and counted by trypan blue exclusion. These cells (CEM-IIIB) were seeded in each well of a 96-well microtiter plate at 5×103 cells per well. Following the addition of cells to each well, the oligonucleotides were added at the indicated concentrations and serial half log dilutions. Infectious HIV-1IIIB was immediately added to each well at a multiplicity of infection determined to give complete cell killing at 6 days post-infection. Following 6 days of incubation at 37° C., an aliquot of supernatant was removed from each well prior to the addition of the tetrazolium dye XTT to each well. The XTT was metabolized to a formazan product by viable cells and the results calculated spectrophotometrically with a Molecular Devices Vmax Plate Reader. ...

example 3

HSV-1 Inhibition HSV-1 Infection ELISA Assay

[0080] Confluent monolayers of human dermal fibroblasts were infected with HSV-1 (KOS) at a multiplicity of 0.05 pfu / cell. After a 90 minute adsorption at 37° C., virus was removed and culture medium containing oligonucleotide at the indicated concentrations was added. Two days after infection medium was removed and cells fixed by addition of 95% ethanol. HSV antigen expression was quantitated using an enzyme linked immunoassay. Primary reactive antibody in the assay was a monoclonal antibody specific for HSV-1 glycoprotein B. Detection was achieved using biotinylated goat anti-mouse IgG as secondary antibody followed by reaction with streptavidin conjugated B-galactosidase. Color was developed by addition of chlorophenol red B-D-galactopyranoside and absorbance at 570 nanometers was measured. Results are expressed as percent of untreated control.

Virus Yield Assay.

[0081] Confluent monolayers of human dermal fibroblasts were infected wi...

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Abstract

Modified oligonucleotides having a conserved G4 sequence and a sufficient number of flanking nucleotides to significantly inhibit the activity of a virus or phospholipase A2 or to modulate the telomere length of a chromosome are provided. G4 quartet oligonucleotide structures are also provided. Methods of prophylaxis, diagnostics and therapeutics for viral-associated diseases and diseases associated with elevated levels of phospholipase A2 are also provided. Methods of modulating telomere length of a chromosome are also provided; modulation of telomere length is believed to play a role in the aging process of a cell and in control of malignant cell growth.

Description

CROSS REFERENCE TO RELATED APPLICATIONS [0001] The present application is a continuation of U.S. application Ser. No. 08 / 403,888 filed Jun. 12, 1995, which is the national phase of PCT Application Serial No. PCT / US93 / 09297 filed Sep. 29, 1993, which is a continuation-in-part of U.S. application Ser. No. 07 / 954,185 filed Sep. 29, 1992, now abandoned, each of which is incorporated herein by reference in its entirety.FIELD OF THE INVENTION [0002] This invention relates to the design and synthesis of oligonucleotides which can be used to inhibit the activity of viruses in vivo or in vitro and to treat viral-associated disease. These compounds can be used either prophylactically or therapeutically for diseases associated with viruses such as HIV, HSV, HCMV and influenza. Oligonucleotides capable of inhibiting phospholipase A2 enzyme activity are also provided which may be useful for the treatment of inflammatory disorders, as well as neurological conditions. Oligonucleotides designed for...

Claims

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Application Information

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IPC IPC(8): A61K48/00A01N43/04C12N15/09A01N63/00A61K31/70A61K38/00A61P31/12A61P31/16A61P35/00C07H21/00C07H21/04C12N15/113C12N15/115C12N15/117C12P19/34C12Q1/68C12Q1/70
CPCA61K31/70A61K38/00C12Y301/01004C07H21/00C12N15/1133C12N15/1137C12N15/115C12N15/117C12N2310/151C12N2310/18C12N2310/315C12N2310/321C12N2310/341C12N2310/346C12Q1/701C12Y207/07049C12N2310/3521A61P31/12A61P31/16A61P31/18A61P31/22A61P35/00C12Q1/70A01N43/04C12Q1/68
Inventor HANECAK, RONNIE C.ANDERSON, KEVIN P.BENNETT, C. FRANKCHIANG, MING-YIBROWN-DRIVER, VICKIE L.ECKER, DAVID J.VICKERS, TIMOTHYWYATT, JACQUELINE R.
Owner IONIS PHARMA INC
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