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Treatment and prevention of influenza

A technology for influenza A virus and nucleic acid constructs, which is applied in the field of nucleic acid constructs and nucleic acid constructs encoding double-stranded RNA molecules, and can solve problems such as high mortality and high pathogenic effects

Inactive Publication Date: 2010-12-15
MAT MALTA ADVANCED TECH +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

But the virus can be highly pathogenic in poultry, with sudden outbreaks leading to high mortality in infected poultry flocks

Method used

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  • Treatment and prevention of influenza
  • Treatment and prevention of influenza
  • Treatment and prevention of influenza

Examples

Experimental program
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Embodiment 1

[0234] Example 1. Selection of shRNA sequences for inclusion in transgenes

[0235] The most highly conserved genes PB1, PB2, PA, NP and M1 of influenza A were selected for RNAi targeting. The inventors used siVirus (a web-based antiviral siRNA design software for highly divergent viral sequences, Naito et al., 2006) to identify highly conserved regions within selected genes and predict siRNAs capable of screening for shRNA selection sequence. The software highlights some regions within the analyzed influenza A genome segments that are of particular interest for shRNA design, namely the 3' regions of PB1, PB2, PA and NP. More specifically, these are segment 1 (PB2 gene) nucleotides 2240-2341; segment 2 (PB1 gene) nucleotides 2257-2341; segment 3 (PA gene) nucleotides 2087-2233; segment 5 (NP gene) nucleotides 1484-1565.

[0236] The inventors selected 29 siRNA sequences predicted from siVirus to screen for shRNA selection (Table 1). Several algorithms are available for s...

Embodiment 2

[0245] Example 2. Chicken promoter sequence

[0246] It is known that when designing transgenic constructs it is desirable to include a promoter that includes upstream sequences to allow efficient attachment of a polymerase to the promoter sequence. Nonetheless, the chicken U6 promoter was designed and tested to contain the minimum amount of promoter sequence required to cause shRNA transcription, thus allowing for a reduction in the overall size of the transgene construct.

[0247]Two versions of the construct, pcU6-4shNP-1496 and pcU6-4(+100)shNP-1496, were tested for shRNA expression by RNAse protection assay, or for viral silencing. The first plasmid contains the minimal chicken U6-4 sequence required to express the shNP-1496 short hairpin RNA. The second plasmid contained 100 bp of additional cU6-4 promoter upstream sequence. It is expected that the second construct containing 100 bp of additional upstream sequence will provide better shRNA expression.

[0248] Table...

Embodiment 3

[0252] Example 3. Construction of ddRNAi plasmids for expressing selected shRNAs

[0253] Chicken polymerase III promoters cU6-1 (GenBank accession number DQ531567), cU6-3 (DQ531569), cU6-4 (DQ531570) and c7SK (EF488955) were used as templates to construct ddRNAi expression of selected shRNAs via one-step PCR Plasmid ( figure 1 ). The PCR for constructing the plasmid uses primers TD135 paired with TD218 or TD275 for the cU6-1 promoter; TD175 paired with TD216, TD274 or TD302 for the cU6-4 promoter; TD176 paired with TD217 for the cU6-3 promoter; TD269 was paired with TD307 or TD316 for the c7SK promoter (see Table 4 for details of primer sequences and specific shRNAs amplified). Reverse primers in each PCR were designed to contain the last 20 nucleotides of each promoter sequence, shRNA sense, loop, and shRNA reaction sequences, and were purified by HPLC. The full-length amplified expression cassette product was ligated into pGEM-T Easy and then sequenced.

[0254] Among...

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Abstract

The present invention relates to nucleic acid molecules comprising a double-stranded region, and nucleic acid constructs encoding therfor, that are useful for the treatment and / or prevention of influenza. In particular, the present invention relates to nucleic acid constructs encoding a double stranded RNA molecule(s) that can be used to produce transgenic poultry, for example chickens, such that they are at least less susceptible to an avian influenza infection. Also provided are nucleic acid molecules comprising a double-stranded region that can be used as a therapeutic to treat and / or prevent, for example, avian influenza in poultry.

Description

field of invention [0001] The present invention relates to nucleic acid molecules comprising double-stranded regions, nucleic acid constructs encoding them, which can be used for treating and / or preventing influenza. In particular, the present invention relates to nucleic acid constructs encoding double-stranded RNA molecules, which can be used to generate transgenic animals, such as chickens, so that they are at least less susceptible to avian influenza infection. Also provided are nucleic acid molecules comprising double stranded regions which can be used as therapeutic agents for the treatment of, for example, avian influenza in poultry. Background of the invention [0002] There are three known types of influenza viruses, A, B and C, which belong to the family of single-stranded negative-sense enveloped RNA viruses called Orthomyxoviridae. The viral genome is approximately 12000-15000 nucleotides in length and contains 8 RNA segments (7 in type C), encoding 11 proteins....

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/44C12N15/63C12N15/85C12N15/90A61P31/16A61K39/145A23L13/00A23L13/50C12N15/113
CPCC12N2830/90C12N2800/90A01K2227/105C12N2830/38C12N2320/11C12N2310/14C12N2310/53A01K2217/058A01K2227/30A01K2267/0337C12N15/1131C12N2760/16111C12N2310/111C12N15/85A61P31/16C12N15/11A01K67/027A61K39/145
Inventor T·J·多兰J·C·麦凯R·J·莫尔J·W·洛温塔尔S·G·蒂亚克
Owner MAT MALTA ADVANCED TECH
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