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Paramyxorividae virus vector defective in envelope gene

A technology of paramyxovirus and virus vector, applied in the direction of virus/bacteriophage, vector, virus, etc., can solve the problems of failure and achieve high gene transfer efficiency

Inactive Publication Date: 2002-06-26
DNAVEC RES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, development of available envelope gene-deficient genome-based vectors has not been successful

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

[0151] [Example 1] Construction of F-deficient Sendai virus

[0152] Construction of F-deficient SeV genome cDNA and plasmid expressing F

[0153] Digestion of Sendai virus (SeV) full-length genomic cDNA, pSeV18, with SphI / KpnI + b(+) (Hasan, M.K. et al., 1997, "Journal of General Virology" 78:2813-2820) ("pSeV18+b(+)" is also called "pSeV18 + ”), reclaim the resulting fragment (14673bp) and clone it into pUC18, which is named as plasmid pUC18 / KS. On this pUC18 / KS, the F-deficient site is constructed. By combining the method of PCR ligation reaction, the defect of the F gene is completed, As a result, the ORF of the F gene (ATG-TGA=1698bp) was removed; therefore, such atgcatgccggcagatga (SEQ ID NO: 1) was ligated thereto at the EcoT22I site to construct the F-deficient SeV genome cDNA (pSeV18 + / ΔF). In PCR, a PCR product generated by using a primer pair (forward: 5'-gttgagtactgcaagagc / SEQ ID NO: 2, reverse: 5'-tttgccggcatgcatgtttcccaaggggagagttttgcaacc / SEQ ID NO: 3) was l...

Embodiment 2

[0165] [Example 2] Confirmation of the function of SeV-F protein expressed by helper cells

[0166] It was tested whether the SeV-F protein induced by the helper cells retained the original protein function.

[0167] After seeding LLC-MK2 / F7 cells in a 6 cm dish and proliferating to confluence, they were infected with adenovirus AxCANCre (as described above) at a multiplicity of infection = 3 according to the method of Saito et al. Then, in an incubator at 37°C, 5% CO 2 Under conditions, the cells were cultured for 3 days in MEM (serum-free) containing trypsin (7.5 [mu]g / ml; GIBCOBRL).

[0168] The culture supernatant was discarded, the cells were washed twice with PBS buffer, scraped off with a spatula, and collected by centrifugation at 1500 xg for 5 minutes. As above (attached image 3 ) was confirmed by Western blot that the protein expressing F was cleaved by trypsin. The SeV-F protein was synthesized as F0, an inactive protein precursor which was then activated after...

Embodiment 3

[0169] [Example 3] Formation of functional RNP with F-deficient genome and virus particles

[0170] In order to recover virus particles from defective viruses, it is necessary to use cells expressing the defective protein. Therefore, when trying to recover the defective virus with cells expressing the defective protein, we found that the expression of the F protein by the helper cell line was quickly stopped due to the vaccinia virus used in the F-deficient SeV (attached Figure 5), thus viral reconstitution based on direct supply of the F protein from a helper cell line failed. It has been reported that vaccinia virus is incapable of replicating, but this can be achieved by treating vaccinia virus with long-wavelength ultraviolet light (long-wavelength UV) in the presence of added psoralen (PLWUV treatment) (Tsung et al., J. Virol. "70, 165-171, 1996), the activity of T7 expression was not weakened. Therefore, virus reconstitution was attempted by using PLWUV-treated vaccin...

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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 paramyxovirus envelope gene defective virus vector. 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 have limitations in gene transfer efficiency and sustained expression, and are also cytotoxic and immunogenic, which are important issues when these vectors are used clinically (Lamb, R.A. and Kolakofsky, D., Viral Field of Science, Paramyxoviridae: Viruses and Their Replication. Third Edition, (B.N. Fields, D.M. Knipe, and P.P. Howley Eds.) pp.1177-1204 (Philadelphia, Lippincott-Raven [1996]). and HSV as countermeasures to this problem, and extensive research is also being carried out to improve existing vectors. However, all of these vectors are in the form of DNA in the nucleus throughout their life cycle. Therefore, it is difficult to completely o...

Claims

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Application Information

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IPC IPC(8): C12N15/09A61K48/00C12N7/01C12N7/04C12N15/45C12N15/86
CPCC12N2760/20222C12N2760/18822A61K2039/5256C12N2760/18843A61K48/00C12N2760/18862C12N2760/18845C12N7/00C12N2810/6081A61K2039/5254C12N2800/30C12N15/86Y02A50/30
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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