Oligonucleotide compound and method for treating nidovirus infections

Inactive Publication Date: 2005-08-04
AVI BIOPHARMA +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Benefits of technology

[0050] For use in inhibiting replication of human SARS virus, the compound may contain one of the sequences SEQ ID NOS: 36-43. For use in inhibiting replication of simian hemorrhagic fever virus, the compound may contain the SEQ ID NOS: 44 and 45.
[0051] In the method of the invention for inhibiting nidovirus replication in virus-infected cells, the cells are exposed to the oligonucleotide compound, in an amount sufficient to inhibit nidovirus replication in the virus-infected cells. The inhibition is due to base-pair binding of the compound to (1) a sequence in a 5′ leader sequence of the nidovirus' positive-strand genomic RNA that includes an internal leader transcriptiona

Problems solved by technology

In addition, coronaviruses cause gastroenteritis and diarrhea in humans and many other serious diseases in non-human animals including mice, chickens, pigs and cats.
The rapid transmission by aerosols and the fecal-oral route and the high mortal

Method used

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  • Oligonucleotide compound and method for treating nidovirus infections
  • Oligonucleotide compound and method for treating nidovirus infections
  • Oligonucleotide compound and method for treating nidovirus infections

Examples

Experimental program
Comparison scheme
Effect test

example 1

Antisense PMO Reduction of SARS Virus and MHV Titer in vitro

[0162] The capability of an antiviral drug to reduce the production of viable virus is a classic measure of antiviral drug activity. The reduction of SARS virus titer produced from SARS-infected Vero-E6 cells cultured in the presence of anti-leader TRS PMO (SEQ ID NOS:26 and 27) was measured and the results shown in FIG. 5. The reduction of MHV titer when MHV-infected Vero-E6 cells were cultured in the presence of a PMO that targets the leader TRS (SEQ ID NO:29) was also determined and is shown in FIG. 9.

[0163] Vero-E6 cells were cultured in DMEM with 10% fetal bovine serum. Vero-E6 cells were plated at approximately 75% confluence in replicate 25 cm2 culture flasks. Cells were rinsed and incubated in 1 ml of complete VP-SFM (virus production serum-free medium, Invitrogen) containing the specified concentration of antisense PMO-P003 conjugate (SEQ ID NOS: 26 or 27) or a PMO-P003 conjugate with an irrelevant sequence (DSsc...

example 2

Antisense PMO Reduction of SARS Plaque Size

[0165] As a separate measure of the antiviral activity of antisense PMO drugs, the anti-leader TRS PMOs were used to measure the reduction of viral replication in a plaque size assay. As viral replication is inhibited a corresponding reduction in the spread of cytopathic effects is observed as a reduction in plaque size. Furthermore, since virus entry and spread are separable phenomena with different criteria, and since some reports indicate that the antisense PMO drugs may inhibit initial virus entry, the plaque size reduction assay is performed. This assay also tests the non-toxicity of drug over longer treatment periods.

[0166] As described above in Example 1, 75% confluent Vero-E6 cells were prepared in plates but not pretreated. Cells were transported to the BSL-3 facility, inoculated with serial dilutions of SARS virus calculated to produce 1, 10 and 100 plaques per plate. The same serial dilution stocks were used for all control and...

example 3

Antisense PMO Reduction of SARS CytoPathic Effects In Vitro

[0167] This assay is a byproduct of the virus titer reduction assay. The observation of cytopathic effects (CPE) is a visual measure of antiviral drug activity. Vero-E6 cells were pretreated, inoculated with SARS virus and cultured as above in the presence of anti-leader TRS PMOs (SEQ ID NOS: 26 & 27). After 24 h, the medium was replaced by fresh complete VP-SFM and cells were incubated a further 24 h at 37 C in the presence of 5% CO2. 48 h after inoculation, the cells were fixed, decontaminated and stained with crystal violet as described in Example 1. CPE is visualized by phase contrast microscopy and recorded with a digital camera as shown in FIG. 6. The data for TRS-1-P003 and TRS-2-P003 correspond to SEQ ID NOS: 26 and 27, respectively. The other treatments presented in FIG. 7 are not relevant to the present invention. From the data presented in FIG. 7, it is clear that anti-leader TRS PMO prevented SARS-induced CPE at...

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Abstract

A method and oligonucleotide compound for inhibiting replication of a nidovirus in virus-infected animal cells are disclosed. The compound (i) has a nuclease-resistant backbone, (ii) is capable of uptake by the infected cells, (iii) contains between 8-25 nucleotide bases, and (iv) has a sequence capable of disrupting base pairing between the transcriptional regulatory sequences in the 5′ leader region of the positive-strand viral genome and negative-strand 3′ subgenomic region. In practicing the method, infected cells are exposed to the compound in an amount effective to inhibit viral replication.

Description

[0001] This patent application claims priority to U.S. Provisional Application No. 60 / 532,701 filed on Dec. 24, 2003, which is incorporated herein in its entirety by reference.FIELD OF THE INVENTION [0002] This invention relates to an oligonucleotide analog for use in treating in animals a coronavirus infection or, more generally, an infection by a member of the Nidovirales order, to an antiviral method employing the analog, and to a method for monitoring binding of the analog to a viral genome target site. REFERENCES [0003] Agrawal, S., S. H. Mayrand, et al. (1990). “Site-specific excision from RNA by RNase H and mixed-phosphate-backbone oligodeoxynucleotides.”Proc Natl Acad Sci USA 87(4): 1401-5. [0004] Allende, R., T. L. Lewis, et al. (1999). “North American and European porcine reproductive and respiratory syndrome viruses differ in non-structural protein coding regions.”J Gen Virol 80 ( Pt 2): 307-15. [0005] Balasuriya, U. B., J. F. Hedges, et al. (2004). “Genetic characterizat...

Claims

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Application Information

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IPC IPC(8): A61K48/00C07F9/6533C07H21/02C07K14/165C12N15/113
CPCC07K14/005C12N15/1131C12N2770/20022C12N2310/3233C12N2310/3513C12N2310/11C07H21/02
Inventor STEIN, DAVID A.BESTWICK, RICHARD K.IVERSEN, PATRICK L.NEUMAN, BENJAMINBUCHMEIER, MICHAEL
Owner AVI BIOPHARMA
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