Paramyxorividae virus vector defective in envelope gene

A paramyxovirus, virus vector technology, applied in the direction of virus/phage, vector, virus, etc., can solve the problem of unsuccessful and achieve the effect of high gene transfer efficiency

Inactive Publication Date: 2007-08-08
DNAVEC RES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the development of available envelope-deficient genome-based vectors has so far been unsuccessful

Method used

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  • Paramyxorividae virus vector defective in envelope gene
  • Paramyxorividae virus vector defective in envelope gene
  • Paramyxorividae virus vector defective in envelope gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0149] [Example 1] Construction of F-deficient Sendai virus

[0150] Construction of F-deficient SeV genome cDNA and plasmid expressing F

[0151] The full-length genomic cDNA of Sendai virus (Sev), pSeV18+b(+) (Hasan, M.K.et al., 1997, J.General Virology 78:2813-2820) was digested with SphI / KpnI ("pSeV18+b(+) "also called "pSeV18+"), the resulting fragment (14673bp) was recovered and cloned into pUC18, which was called plasmid pUC18 / KS. Its F-deficient site was constructed on PUC18 / KS. Combining the PCR-ligation method for F gene deficiency, the result was that the ORF of the F gene was removed (ATG-TGA=1698bp); and ligated with atgcatgccggcagatga (SEQ ID NO: 1) to construct the F-deficient SeV genome cDNA (pSeV18+ / ΔF). In the PCR, the PCR product upstream of the F gene generated by the primer pair (forward: 5'-gttgagtactgcaagagc / SEQ ID NO: 2, reverse: 5'-tttgccggcatgcatgtttcccaaggggagagttttgcaacc / SEQ ID NO: 3) was ligated with EcoT221 to the On the PCR product downstream...

Embodiment 2

[0163] [Example 2] Confirmation of the function of SeV-F protein expressed by helper cells

[0164] To test whether the SeV-F protein induced by helper cells maintains the original function of the protein.

[0165] After LLC-MK2 / F7 was placed in a 6 cm dish and grown to confluence, cells were infected with adenovirus AxCANCre at moi=3 according to the method of Saito et al. Then in MEM (serum-free) containing adenase (7.5 μg / ml; GIBCOBRL) at 37°C and 5% CO 2 The cells were cultured under gas for three days.

[0166] The culture supernatant was discarded and the cells were washed twice with PBS buffer, detached with a scraper and centrifuged at 1500xg for five minutes to collect the cells. Cleavage of expressed F protein was verified by Western blotting as described above (Fig. 3). SeV-F is first synthesized from F0 as an inactive protein precursor, and then activated by cleavage into two subunits F1 and F2 under the action of trypsin. After induction of F protein expressio...

Embodiment 3

[0167] [Example 3] Formation of functional RNP and virus particles containing F-deficient genome

[0168] When recovering viral particles of a defective virus, it is necessary to use cells expressing the defective protein. However, when cells expressing the defective protein were used to recover the defective virus, it was found that the expression of the F protein using the helper cell line was rapidly terminated due to the vaccinia virus used in the reconstitution of the F-deficient SeV (Fig. Reconstitution of helper cell lines directly supplied with F protein-based viruses failed. Treatment of vaccinia virus with long-wavelength ultraviolet light (long-wave UV) in the presence of psoralen (PLWUV treatment) has been reported to inactivate the replication capacity of vaccinia virus without compromising the activity of T7 expression (Tsung et al., J Virol 70, 165-171, 1996). Therefore, viral reconstitution was attempted using PLWUV-treated vaccinia virus (PLWUV-VacT7). Ultr...

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Abstract

Virus virions defective in F gene are successfully collected by using a Sendai virus genomic cDNA with deletion of F gene. Further, infectious viral particles defective in F gene are successfully constructed by using F-expression cells as helper cells. Also, virus virions defective in F gene and HN gene are successfully collected by using a virus genomic cDNA with deletion of both of F gene and HN gene. Further, infectious viral particles defective in F gene and HN gene are successfully produced by using F- and HN-expression cells as helper cells. A virus being defective in F gene and HN gene and having F protein is constructed by using F-expression cells as helper cells. Further, a VSV-G pseudo type virus is successfully constructed by using VSV-G-expression cells. Techniques for constructing these defective viruses contribute to the development of vectors of Paramyxoviridae usable in gene therapy.

Description

technical field [0001] The invention relates to a viral vector of enveloped gene-deficient paramyxovirus. Background technique [0002] Viral vectors derived from retroviruses, adenoviruses, and adeno-associated viruses have been used so far in many clinical approaches to gene therapy. These gene therapy vectors also have certain limitations in gene introduction efficiency and sustained expression, and also have cytotoxicity and immunogenicity, so when these vectors are applied clinically, there are also larger problems (Lamb, R.A. and Kolakofsky, D., Fields of Virology, Paramyxoviridae: Viruses and Their Replication. Third Edition, (eds. B.N. Fields, D.M. Knipe, and P.P. Howley) pp. 1177-1204 (Philadelphia, Lippincott-Raven [1996]). Proposed Development of new vectors using lentiviruses and HSVs as a countermeasure to this problem, and extensive research is underway to improve existing vectors. However, all of these vectors exist as DNA in the nucleus during their life cyc...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/86C12N15/45C12N7/01C12N5/10C12N15/09A61K48/00C12N7/04
CPCC12N2760/20222C12N2760/18822A61K2039/5256C12N2760/18843A61K48/00C12N2760/18845C12N2760/18862C12N7/00A61K2039/5254C12N2810/6081C12N2800/30C12N15/86Y02A50/30
Inventor 北里海雄朱亚峰上田泰次长谷川护饭田章博时任文乃平田隆洋德炭刚隈秀和浅川诚
Owner DNAVEC RES
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