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Attenuated minus-stranded rna virus

An RNA virus, negative-strand technology, applied in the field of attenuated negative-strand RNA viruses, can solve problems such as limitations

Inactive Publication Date: 2010-02-10
DNAVEC CORP
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Compared with carrying the gene on the genome for transcription, although the number of molecules can be greatly reduced, this method still has limitations in terms of reducing cytotoxicity and immune response

Method used

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  • Attenuated minus-stranded rna virus
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  • Attenuated minus-stranded rna virus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0173] [Example 1] Gene

[0174] 1-1. Sendai virus

[0175] Sendai virus uses Z strain 15kb, makes it delete each gene, and inserts amino acid mutation.

[0176] 1-2. Reporter gene

[0177] EGFP: The green fluorescent protein, 720b (Accession No. U57606), obtained by improving the nucleotide sequence derived from luminescent jellyfish, was loaded into the Sendai virus genome.

[0178] LacZ: loading of β-galactosidase, 3.1 kb (Accession No. U13184) into Sendai virus genome.

Embodiment 2

[0179] [Example 2] Cell culture

[0180] 2-1. Cell lines

[0181] LLC-MK2 Cell line derived from macaque kidney

[0182] CV-1 cell line derived from African green monkey kidney

[0183] HEK 293 Cell line derived from human fetal kidney

[0184] Mesenchymal cells derived from mouse bone marrow were obtained from the thigh of C57BL / 6 mice

[0185] 2-2. Cell culture

[0186] Cell lines LLC-MK2 (ATCC CCL-7) and CV-1 (ATCCCCL-70) derived from monkey kidney were suspended in 10% fetal bovine serum (fetal bovine serum; FBS, GIBCO-BRL, Cat.No. 10099-141), 100 μg / ml penicillin / 100 units / ml streptomycin (Nakarai Tesque, Cat.No.26253-84) minimum essential medium (Minimal essential medium) (MEM) (Invitrogen-GIBCO, Cat.No .11095-080) culture medium, cultivated under the conditions of 5% carbon dioxide and 37°C. HEK 293 cell line (ATCC CRL-1573) was suspended in Dulbecco's Modified Eagle Medium (DMEM) (Invitrogen-GIBCO , Cat.No.11995-065) culture medium, cultivated under the same con...

Embodiment 3

[0187] [Example 3] modulation virus

[0188] 3-1. Modulation of Sendai virus vector

[0189] The packaging cell line LLC-MK2 / F7 (F protein expression cell) (Li, H.O., Zhu, Y.F., Asakawa, M., Kuma, H., Hirata, T., Ueda, Y., Lee, Y.S., Fukumura, M., Iida, A., Kato, A., et al. (2000), J Virol 74, p6564-6569.), LLC-MK2 / F7 / M62 (M Protein expression cells) (Inoue, M., Tokusumi, Y., Ban, H., Kanaya, T., Shirakura, M., Tokusumi, T., Hirata, T., Nagai, Y., Iida, A., and Hasegawa, M. (2003), J Virol 77, p6419-6429.), recovered F gene-deleted vectors (SeV / ΔF) and M gene-deleted vectors (SeV / ΔM).

[0190] 3-2. Modulation of adenoviral vector

[0191] The protein encoded by each deleted gene was induced with an adenovirus vector expressing Cre recombinase (AxCANCre) (Nakano, 2003, P147). The LacZ-expressing type 5 adenoviral vector (AdenoCALacZ) was recovered from HEK 293 cells and passed through Adeno-X TM Rapid Titer Kit (BD Bioscience, Cat. No. K1653-1) was used to measure the tit...

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Abstract

Disclosed is attenuated minus-stranded RNA virus. It is found that the mutation of an amino acid residue at position-1214 in the amino acid sequence for Sendai virus L-protein (Y1214F) can inhibit thegenome replication activity and / or the genome transcription activity of the virus. It is also found that a cytotoxicity or immunological response can be reduced compared with the original one by deleting a specific gene from a viral genome.

Description

technical field [0001] The present invention relates to an attenuated negative-strand RNA virus. Background technique [0002] After the reverse genetics technology was developed in 1994, virus molecules of infectious particles can be generated in vitro (in vitro) by using the cDNA of a virus with a negative-strand RNA genome as a template. This technology makes it possible to modify the viral cDNA arbitrarily. At present, a non-segmented negative-strand RNA viral vector (minus-strand non-segmented RNA viral vector) has been developed as a variety of gene transfer vectors (see Non-Patent Document 1 ). [0003] An important condition for viral vectors to be applied to humans is attenuation. There are generally two methods for virus attenuation. One approach is to remove genes from the viral genome. Examples of viruses that cause respiratory disease in young children include human metapneumovirus (HMPV) and respiratory syncytial virus (RSV). Infants under 2 years old are ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/09C12N7/00C07K14/08C07K14/115
CPCC12N2760/18822C12N7/00A61K2039/5254C12N2760/18861C07K14/005A61K48/00C07K14/08C07K14/115C12N15/09
Inventor 吉崎真理子井上诚长谷川护
Owner DNAVEC CORP
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