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Polynucleotides for Reducing Respiratory Syncytial Virus Gene Expression

a technology of respiratory syncytial virus and polynucleotide, which is applied in the field of polynucleotides for reducing respiratory syncytial virus gene expression, can solve the problems of no specific antiviral treatment licensed for public health use to prevent diseases associated with rsv infection, and no specific antiviral treatment available. , to achieve the effect of reducing the expression of one or more rsv genes, reducing

Inactive Publication Date: 2007-10-11
UNIV OF SOUTH FLORIDA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The present invention provides a method for reducing the expression of respiratory syncytial virus (RSV) genes in a subject by administering polynucleotides that target specific nucleotide sequences within the RSV genes. The polynucleotides may be administered locally or systemically to the airway cells of the subject. The method can be used to treat RSV infections in humans and non-human subjects. The polynucleotides may be double-stranded RNA (dsRNA) or single-stranded RNA (ssRNA) that interferes with expression of the RSV gene in a sequence-specific manner, or an antisense nucleic acid sequence that binds to the RSV mRNA and inhibits translation. The polynucleotides can also be designed to have enzymatic activity and inhibit expression of the target RSV gene.

Problems solved by technology

Previous RSV infection does not prevent subsequent infections, even in sequential years (Bartz, H. et al.
To date, there are no specific antiviral treatments available.
Although many different approaches are being taken to develop prophylactic vaccines, none have been licensed for public health use to prevent diseases associated with RSV infection.
Such triple-stranded structures are unstable and form only transiently under physiological conditions.

Method used

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  • Polynucleotides for Reducing Respiratory Syncytial Virus Gene Expression
  • Polynucleotides for Reducing Respiratory Syncytial Virus Gene Expression
  • Polynucleotides for Reducing Respiratory Syncytial Virus Gene Expression

Examples

Experimental program
Comparison scheme
Effect test

example 1

siNS1 Inhibition of rgRSV Infection

[0146] Two different siRNA oligos for RSV NS1, siNS1 and siNS1a, HPV18E7 (siE7) and Influenza virus PB2 (siPB2) were designed and cloned into the pSMWZ-1 vector (Zhang, W. et al. Genetic Vaccines Ther., 2004, 2:8-12). Analysis of EGFP expression in A549 cells co-transfected with pEGFP and siNS1 / 1a, siE7 or siPB2 demonstrates that none of siRNAs silence the EGFP gene (data not shown). Immunoblotting results show that pre-transfection of A549 cells with siNS1, but not siE7, significantly reduces the expression of NS1 proteins (FIG. 1A), but not that of other viral proteins (data not shown). To test whether siNS1 attenuates virus infection, A549 cells and type-1 IFN deficient (Mosca, J. D. and Pitha, P. M. Mol. Cell. Biol., 1986, 6:2279-2283) Vero cells were transfected with the siNS1, siNS1a, or control siRNAs, and then infected with rgRSV (Hallak, L. K. et al. Virology, 2000, 271:264-275). The results of flow cytometry show a significant decrease i...

example 2

Mechanism of siNS1-Mediated Upregulation of Type-1 IFN Pathway

[0147] The finding that RSV infection of A549 cells, but not Vero cells, is affected by siNS treatment suggests a role of NS1 protein in the promotion of RSV infection by inhibiting the type-1 IFN pathway.

[0148] To verify whether NS1 decreases the amount of type-1 IFN, the expression of IFN-β was examined by immunoblotting. The results show that A549 cells transfected with siNS1 or siNS1a, upon RSV infection, produce significantly increased amounts of IFN-β, compared to the different controls, including totally unrelated siRNA with no homology to mammalian genes (siUR), (FIGS. 2A and 2B).

[0149] To further examine the role of NS1 in regulating the IFN pathway, RNAs from control and siNS1-transduced cells were isolated and subjected to microarray analyses. The results show that siNS1 treatment increased the expression (≧6 fold-change) of 25 IFN-inducible genes compared to rgRSV infection alone (Table 1), and the expressi...

example 3

Silencing NS1 Polarizes Human Dendritic Cells Toward a Th1-Promoting Phenotype

[0151] Monocytes isolated from human peripheral blood were cultured with requisite cytokines to test whether siNS1 expression affects RSV-infected DC activity. Thus, the IFN-α and IFN-β concentrations were measured in the supernatants from cultured, infected, monocyte-derived DCs transfected with siNS1 or control. The data show that siNS1 treatment induces a significantly higher production of both type-1 IFNs in infected DCs than it does in controls (FIG. 3A). Furthermore, to assess the effect of siNS1-treated DCs on T-cell function, allogenic naïve CD4+T cells were co-cultured with RSV-infected DCs treated with or without siNS1. The results of intracellular cytokine staining showed an increase in IFN-γ and a decrease in IL-4 secretion in naïve CD4+T cells for siNS1-treated, RSV-infected DCs, compared with controls (FIG. 3B).

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Abstract

This invention pertains to polynucleotides, such as small interfering RNA (siRNA), useful for reducing the expression of respiratory syncytial virus (RSV) genes within a subject; and methods for treating a patient suffering from, or at risk of developing, an RSV infection by administering such polynucleotides to the subject.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This application claims benefit of U.S. Provisional Application Ser. No. 60 / 481,738, filed Dec. 4, 2003, and U.S. Provisional Application Ser. No. 60 / 522,180, filed Aug. 26, 2004, each of which is hereby incorporated by reference herein in its entirety, including any figures, tables, nucleic acid sequences, amino acid sequences, and drawings.BACKGROUND OF THE INVENTION [0002] Respiratory syncytial virus (RSV) a major viral respiratory pathogen and is the leading cause of lower respiratory tract infection in infant, young children and the elderly with immunocompromise (Collins, P. L. et al Respiratory syncytial virus. In: D. M. Knipe, P. M. Howley and D. E. Griffin, Editors, 4th ed., Fields Virology Vol. 1, Lippincott-Raven, Philadelphia, 2001, pp. 1443-1485), and is also a risk factor for the development of asthma (Behera, A. K. et al. J Biol Chem, 2002, 277:25601-25608). RSV produces an annual epidemic of respiratory illness, causing b...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): A61K31/70C07H21/02C07H21/04C12N15/113
CPCA61K31/70C07H21/02C07H21/04A61K31/713C12N2310/111C12N2310/14C12N15/1131
Inventor MOHAPATRA, SHYAM S.ZHANG, WEIDONG
Owner UNIV OF SOUTH FLORIDA
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