Chemistry: molecular biology and microbiology – Virus or bacteriophage – except for viral vector or...
Patent
1998-03-31
1999-12-14
Budens, Robert D.
Chemistry: molecular biology and microbiology
Virus or bacteriophage, except for viral vector or...
435465, 435467, 435236, 435239, 435325, 435352, 435354, C12N 700, C12N 702, C12N 500
Patent
active
060016337
DESCRIPTION:
BRIEF SUMMARY
FIELD OF THE INVENTION
The present invention relates to cells producing a recombinant retrovirus.
BACKGROUND ART
A method for introducing a retrovirus as a vector into higher animal cells has excellent features such as less cytotoxicity associated with gene transfer and integration of the transferred gene into the chromosome with stable maintenance. For these reasons, this method has not only been used for experimental gene transfer but for gene therapy as well. The retrovirus used as a vector is mainly derived from the Moloney Murine Leukemia Virus (MoMuLV), but a retrovirus derived from the Avian Leukosis Virus (ALV) has also been developed. These retroviruses have a single-strand RNA as a genome, on which gag, pol and env genes coding for proteins necessary for virus construction are located. The viral RNA genome is coated by capsid proteins and further coated by matrix proteins, transmembrane proteins and surface proteins. These viral coating proteins are encoded by the gag and env genes. Further, the pol gene codes for reverse transcriptase, protease and integrase which are important in the viral life cycle.
When cells are infected with such a retrovirus, the viral RNA is transcribed into DNA by reverse transcriptase, and the resultant DNA is integrated into a cellular chromosome by integrase to make a provirus. The gag, pol and env genes are expressed by this provirus to construct a viral particle and an RNA viral genome is produced at the same time. This viral RNA is then encapsidated into the viral particle to replicate the virus. When produced from the cell, this virus can infect other cells.
In order to use this retrovirus as a vector, it is necessary to prepare a "recombinant retrovirus" into which a foreign gene has been introduced. A fundamental method for preparing a recombinant retrovirus has been established, in which the recombinant retrovirus is prepared using a particular cell called a "packaging cell".
In a packaging cell, a retrovirus genome having the gag, pol and env genes is integrated into a chromosome, and all the proteins necessary to construct a viral particle are expressed by the integrated genes. A mutation has been introduced in said retrovirus-derived genome. Namely, the .psi. region, which is necessary to encapsidate the virus genome into the viral particle, is deleted, and thus the RNA genome cannot be encapsidated into the viral particle. When a plasmid which have the .psi. region, two long terminal repeat regions (LTRs) (generally, they are 5' LTR and 3' LTR, where the initiation region of reverse transcription and promoter and enhancer sequences are located in 5' LTR, and a poly A addition signal is located in 3' LTR), and the foreign gene franked between two LTRs, are introduced into a packaging cell line, the RNA genome derived from this plasmid is encapsidated into the viral particle. A retrovirus vector having a foreign gene is thus produced. This recombinant retrovirus infects a target cell and integrates the foreign gene into the chromosome of the infected cell, wherein said foreign gene can stably be expressed in the target cell.
In introducing a gene into a packaging cell, the greater the production of retrovirus by the cell, that is, the higher the viral titer, the greater the number of cells into which said gene is introduced. Introduction of a gene into a large number of cells facilitates experiments for gene transfer and is important for efficient gene therapy.
In order to increase the viral titer, recombinant plasmids DOL and DOL.sup.- have been constructed, which have the Polyoma virus early region gene and the replication origin containing the promoter of this early region gene (A. J. Korman, J. D. Frantz, J. L. Strominger and R. C. Mulligan: Expression of human class II major histocompatibility complex antigens using retrovirus vectors. Proc. Natl. Acad. Sci. USA, 84, 2150-2154 (1987)). These recombinant plasmids are a case to which the Polyoma virus early region gene and its replication system are applied.
The polyoma virus is a circular DNA virus i
REFERENCES:
Korman et al., Proc. Natl. Acad. Sci. USA 84:2150-2154, Apr. 1987.
Yoshimatsu et al., Hum. Gene Ther. 9:161-172, Jan. 20, 1998.
Ikenaka Kazuhiro
Yoshimatsu Tadanori
Budens Robert D.
Wakunaga Seiyaku Kabushiki Kaisha
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