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Non-replicating paramyxoviridae virus vector

A technology of paramyxoviruses and vectors, applied in the field of non-replicating paramyxoviridae virus vectors, capable of solving problems such as preparation of missing L genes

Inactive Publication Date: 2009-08-26
DNAVEC CORP
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, in the case of Paramyxoviridae viral vectors containing SeV vectors, there have been no reports indicating the preparation of vectors lacking the L gene.

Method used

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  • Non-replicating paramyxoviridae virus vector
  • Non-replicating paramyxoviridae virus vector
  • Non-replicating paramyxoviridae virus vector

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0179] [Example 1] The influence of the C protein of SeV on the production efficiency of MiniSeV / GFP / M / F / HN

[0180] Using Lipofectamine 2000 reagent, introduce pCAGGS-NP(Z), pCAGGS-P(Z) / 4C(-), pCAGGS-L(TDK) and pCI control or pCI-4C / P(-) into 2e5 cells the day before / well (12-well collagen-coated plate, IWAKI) in BHK-21 cells seeded. The next day, after infection with MiniSeV / GFP / M / F / HN as virus species, virus production was carried out with VP-SFM culture solution containing trypsin (final concentration: 2.5 μg / ml). GFP fluorescence pictures of production cells were taken on day 4 after infection. The culture supernatant on day 4 was recovered and infected into fresh LLC-MK2 cells (24-well plate), and GFP fluorescence pictures were taken 3 days later.

[0181] Compared with the condition of introducing a control plasmid [pCI-neo] that does not express protein C, a higher virus production efficiency was obtained under the condition of introducing the protein C expression p...

Embodiment 2

[0182] [Example 2] Production of MiniSeV / GFP / M / F / HN

[0183] Using Lipofectamine 2000 reagent, pCAGGS-NP(Z), P(Z) / 4C(-), L(TDK) and pCI-4C / P(-) were introduced into the previous day with 8e5 cells / well (6-well collagen coated plate, IWAKI) inoculated BHK-21 cells. The next day, after infection with MiniSeV / GFP / M / F / HN as virus species, virus production was carried out with VP-SFM culture solution containing trypsin (final concentration: 2.5 μg / ml). From day 1 to day 7 after infection, GFP fluorescence photos were taken every day, the culture supernatant was recovered, and the culture medium was replaced. The CIU measurement was performed on the culture supernatant collected on days 1 to 7.

[0184] Such as Figure 4 As shown, MiniSeV / GFP / M / F / HN with a high titer above 1e7GFP-CIU / ml was recovered from the culture supernatant on days 3-6.

Embodiment 3

[0185] [Example 3] In vitro expression of MiniSeV / GFP / M / F / HN

[0186] MiniSeV / GFP / M / F / HN was infected at MOI=100, 10, 1 into HeLa cells seeded with 3e5 cells / well (12-well collagen-coated plate, IWAKI) on the same day. And MiniSeV / GFP / M / F / HN was co-infected with MOI=1 and SeVagp55 / dF with MOI=5. GFP photographs were taken on days 1 and 2 post-infection.

[0187] Such as Figure 5 As shown, when MiniSeV / GFP / M / F / HN was infected with MOI=1 alone, only weak GFP expression was observed, and when infected with MOI=100, it was confirmed that it was close to Helper-SeV (SeVagp55 / ΔF) co-infection ( GFP expression at levels equivalent to replicating SeV).

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Abstract

The present inventors succeeded in producing non-replicating SeV vectors whose genomic RNAs lack all genes for the NP, P, and L proteins, which are RNP-constituting proteins. The present inventors confirmed that the NP / P / L-deficient SeV vectors carrying a marker gene such as GFP provide high productivity, and high transfer and expression efficiencies of foreign genes (high MOI infection is essential for achieving high expression levels). By lacking the L gene or two or more of the NP, P, and L genes, the vectors of the present invention enable lowering the level of virus-derived proteins expressed in host cells, thereby reducing the immunogenicity upon in vivo administration.

Description

technical field [0001] The invention relates to a non-replicating Paramyxoviridae virus vector. Background technique [0002] The Sendai virus vector (SeV vector) is a cytoplasmic viral vector (the entire process of expression is completed in the cytoplasm), so there is no need to worry about genetic toxicity caused by gene integration into the host chromosome when used in vivo. In addition, it has several superior properties, such as high-efficiency gene introduction and expression in vitro and in vivo, and long-term sustained expression in vitro. Therefore, in the application of gene therapy, gene vaccine or antibody production and functional analysis, SeV is expected to be more widely used as a gene transfer carrier (Non-Patent Documents 1 and 2). [0003] However, when using the Sendai virus vector to express foreign genes, the viral structural proteins (especially the RNP constituent proteins NP, P, and L proteins required for transcription and replication) are also ex...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/09A61K48/00C12N7/00C12N5/10
CPCC12N15/86C12N2820/00C12N2760/18843C12N2760/18871A61K48/00C12N7/00C12N15/09
Inventor 游军井上诚长谷川护
Owner DNAVEC CORP