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Inhibition of viral gene expression using small interfering RNA

A technology of small interference and expression vectors, applied in the direction of positive-sense single-stranded RNA viruses, viruses, DNA/RNA fragments, etc., can solve the problem of incomplete treatment and other problems

Inactive Publication Date: 2012-09-26
SOMAGENICS INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] Because existing treatments are not fully effective (reviewed in [4, 12]), attempts have been made to discover potent nucleic acid-based inhibitors against HCV

Method used

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  • Inhibition of viral gene expression using small interfering RNA
  • Inhibition of viral gene expression using small interfering RNA
  • Inhibition of viral gene expression using small interfering RNA

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0121] Example 1: Design and Construction of shRNA Expression Cassette, T7 Transcription Reaction and Reporter Gene Test

[0122] Chemically synthesized oligonucleotides were obtained from IDT (Coralville, IA), resuspended in RNase- and pyrogen-free water (Biowhittaker), and annealed as described below. The following oligonucleotide pairs used to prepare shRNAs contain the T7 promoter element (double underlined), sense and antisense HCVIRES sequences, and the miR-23 small RNA loop structure (reported to favor cytoplasmic localization [21，22] ).

[0123] T7-HCVa-wt fw:

[0124] 5'- agcacgaatcctaaa c ctca

[0125] aagaCTTCCTGTCAtctttgaggtttaggatt c gtgctcTT-3' (SEQ ID NO: 1);

[0126] T7-HCVa-wt rev:

[0127] 5’-AAgagcacgaatcctaaa c ctcaaagaTGACAGGAA

[0128] Gtctttgaggtttaggatt c gtgct -3'

[0129] (SEQ ID NO: 2)

[0130] (T7 promoter sequence is double underlined). T7 transcripts of HCVa-mut shRNA were identical except for the nucleotide changes (G→C vs. C→G)...

Embodiment 2

[0146] Example 2: shRNA Inhibition of HCV IRES-mediated Gene Expression in Human Tissue Culture Cells system

[0147] In this study, the ability of short interfering RNA (shRNA and siRNA) targeting the conserved region of hepatitis C IRES designed and constructed according to Example 1 to inhibit HCVIRES-mediated reporter gene expression in human tissue culture cells was tested.

[0148] Figure 1A Shows the HCV IRES target site (Panel A) and the HCV shRNA transcribed via T7 from a template prepared from a hybrid oligonucleotide containing the T7 promoter sequence and the HCV IRES target ( Figure 1B ). The underlined residues are those that were changed to generate the mutant HCV shRNA. This shRNA contains the miR-23 small RNA loop structure previously suggested to facilitate cytoplasmic localization [21，22] and 25 base pairs of RNA stem and 2 nucleotides at the 5' (2 guanines) and 3' (2 uridines) ends (which can also be base-paired by non-Watson-Crick G:U to hybridiz...

Embodiment 3

[0159] Example 3: shRNA Inhibition of HCV IRES-Mediated Gene Expression in a Mouse Model System

[0160] The ability of HCV shRNA and HCV shRNA expression plasmids to repress target gene expression was extended to a mouse model system by using hydrodynamic injection to deliver these nucleic acids to the mouse liver. Figure 5 shows the injection of a large volume of PBS (1.8 ml) containing pHCV dual Luc, pSEAP2, and shRNA (based on the mass of shRNA or pol III expression vector expressing shRNA, 10-fold excess to target) into the mouse tail vein (n = 4-5 mice). in such as Figure 5B At the indicated time points, luciferin was injected intraperitoneally and mice were photographed with a high-sensitivity, cooled CCD camera. ( Figure 5A Representative mice selected from each group (4-5 mice per group) at the 84 hour time point are shown). HCV shRNA strongly suppressed luciferase expression at all time points tested, from 98% (84-hour time point) to 94% (48-hour time point)...

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Abstract

The invention provides methods, compositions, and kits comprising small interfering RNA (shRNA or siRNA) that are useful for inhibition of viral-mediated gene expression. Small interfering RNAs as described herein can be used in methods of treatment of HCV infection. ShRNA and siRNA constructs targeting the internal ribosome entry site (IRES) sequence of HCV are described.

Description

[0001] Cross-References to Related Applications [0002] This application claims the benefit of PCT Application PCT / US2005 / 032768 filed September 12, 2005, which claims filing on September 10, 2004 pursuant to 35 U.S.C. § 119 (35 U.S.C. § 119) Priority to US Provisional Application No. 60 / 608,574, both of which are hereby incorporated by reference in their entirety. [0003] Statement Regarding Federally Sponsored Research or Development [0004] The present invention was completed in part under the support of the grant number 5R43AI056611 of the National Institutes of Health (National Institutes of Health) in the research stage. The US Government has certain rights in this invention. field of invention [0005] The present invention relates to the use of small interfering RNA (shRNA and siRNA) to inhibit viral gene expression, such as hepatitis C IRES-mediated gene expression. Background of the invention [0006] Treatment and prevention of hepatitis C virus (HCV) infect...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/11A61P31/14A61K31/713C12N15/113
CPCC12N2770/36111C12N2770/24211C12N15/1131C12N2310/14C12N2310/53C12N2310/111A61P31/12A61P31/14A61K31/713C12N15/113
Inventor R·L·卡斯帕尔H·伊尔韦斯A·A·塞伊汗A·V·弗拉索夫B·H·约翰斯顿
Owner SOMAGENICS INC
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