Chemistry: molecular biology and microbiology – Process of mutation – cell fusion – or genetic modification – Introduction of a polynucleotide molecule into or...
Reexamination Certificate
1998-11-03
2001-09-18
Wortman, Donna C. (Department: 1648)
Chemistry: molecular biology and microbiology
Process of mutation, cell fusion, or genetic modification
Introduction of a polynucleotide molecule into or...
C435S320100, C435S465000, C435S235100
Reexamination Certificate
active
06291246
ABSTRACT:
BACKGROUND OF THE INVENTION
(i) Field of the Invention
The present invention relates to a method for the preparation of DNA virus vectors capable of replicating in eukaryotic as well as in prokaryotic cells, and to the DNA virus vectors prepared by said method as well as to cells containing said vectors.
(ii) Description of Related Art
Epstein-Barr virus is one of several herpes viruses in humans. The DNA sequence of the EBV B95.8 isolate has been determined (Baer et al., 1984), and detailed scientific evidence has been worked out mainly with respect to DNA elements which play important roles in the two EBV phases. In the so-called ‘latent phase’ the virus establishes a stable host cell relation during which the vitality of the cell remains unaffected, however, the viral DNA genome is replicated in the form of an extrachromosomal plasmid in the host cells and passed on into the daughter cells. The latent phase may be associated with a transformation or immortalization, respectively, of the cells infected in a latent manner. The replication origin of the plasmid, oriP, is the DNA element essential for maintenance and replication of the EBV genome in the latent phase. (Yates et al., 1985). This DNA element is also active in recombinant plasmids and has been used as such in several ways.
In the so-called ‘lytic or productive phase’ the virus is produced in active manner which in addition to the expression of almost all of the viral proteins necessary for regulation of expression or for expression of structural viral components involves two other DNA elements of the virus: the lytic origin of replication, oriLyt, is responsible for viral DNA amplification (Hammerschmidt and Sugden, 1988), the terminal repeats, TR, are packaging signals indispensable for encapsidation of the amplified EBV DNA (Hammerschmidt and Sugden, 1989).
There is a broad interest in the genetic analysis of EBV functions as well as the construction of recombinant EBV genomes. The interest is based on the fact that EBV genomes may bear additional therapeutical genes or that certain undesired properties or genes of EBV may be removed. The techniques used so far in the alteration of the EBV genome are based on homologous recombination events of recombinant
E. coli
plasmids with EBV genomes in cell lines infected by EBV in the latent manner (Cohen et al., 1989; Hammerschmidt and Sugden, 1989). Alternatively, so-called mini EBV genomes may be prepared in
E. coli
which, however, only contain the viral gene functions important for the latent phase of EBV (Sugden and Hammerschmidt, 1993; Hammerschmidt and Sugden, 1995; Kempkes et al., 1995a; Kempkes et al., 1995b).
DISADVANTAGES OF THE RELATED ART
Up to now it has been impossible to alter or to delete any gene of DNA viruses ≧100 kbp, for example of EBV, in a targeted manner and with high efficiency. Thus, homologous recombination events between an EBV segment cloned in
E. coli
and the endogenous EBV present in cell lines infected in a latent manner are inaccurate, difficult to control and must be rendered detectable by means of co-transfected marker segments or selectable genes. In the present prior art, such approaches are not universally applicable, are slow and do not enable the alteration of genes or genomic segments of DNA viruses ≧100 kbp of EBV, such as those necessary for the maintenance of the EBV virus latent phase in the infected cells.
SUMMARY AND OBJECTS OF THE INVENTION
It is an object of the present invention to provide a method for the preparation of DNA virus vectors capable of replication in eukaryotic as well as in prokaryotic cells which enables the preparation of recombinant DNA viruses having a genome ≧100 kbp.
Thus, according to the invention the disadvantages of the prior art have been overcome by cloning the entire DNA virus genome of viruses having a size of ≧100 kbp in the form of a functional unit. A prerequisite of the cloning e.g. in
E. coli
as a recombinant molecule is the ability to integrate a so-called prokaryotic gene segment into the intact DNA virus genome. Integration of this segment containing replicational functions and a selectable marker for
E. coli
ensures that said recombinant molecule of a size of more than 100 kbp may be transfected e.g. into
E. coli
and stably replicated therein. In addition, this event ensures that a modification of any DNA virus segment e.g. in
E. coli
will be possible by means of conventional recombinant DNA technologies.
REFERENCES:
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patent: 5348886 (1994-09-01), Lee et al.
patent: 19733364 (1999-02-01), None
patent: 694613 (1996-01-01), None
Delecluse et al., Propagation and recovery of intact, infectious Epstein-Barr virus from prokaryotic to human cells. Proc. Natl. Acad. Sci. USA 95:8425-8520, 1998.
Messerle et al., Cloning and mutagenesis of herpesvirus genome as an infectious bacterial artificial chromosome. Proc. Natl. Acad. Sci. USA 94:14759-14763, 1997.
Shizuya et al., Cloning and Stable maintenance of 300-kilobase-pair fragments of human DNA inEscherichia coliusing an F-factor-based vector.
Krisky, et al., “Rapid Method for Construction of Recombinant HSV Gene Transfer Vectors”,Gene Therapy(1997) vol. 4, pp. 1120-1125.
O'Connor et al., “Construction of Large DNA Segements inEscherichia coli”,Science, (1989), vol. 4, p. 1307-1312.
Kempkes et al, “Immortalization of Human Primary B Lmphocytes in vitro with DNA”,Proc. Natl. Acad. Sci., USA, Jun. 1995, pp. 5878-5879.
Kempkes et al., “Immortalization of Human B Lymphocytes by a Plasmid Containing 712 Kilobase Pairs of Epstein-Barr Virus DNA”,Journal of Virology, Jan. 1995, pp. 231-238.
Baer et al., “DNA Sequence and Expression of The B95-8 Epstein-Barr Virus Genome”,Nature, vol. 310, Jul. 1984, pp. 207-211.
Cherepanov et al., “Gene Disruption inEscherichia coli: TcRand KmRcassettes with the option of Flp-catalyzed excision of the antibiotic-resistance determinant”,Gene 08901, 1995, pp. 9-14.
Cohen et al., “Epstein-Barr Virus Nuclear Protein 2 is a Key Determinant of Lymphocyte Transformation”,Proc. Natl. Acad. Sci, USA, vol. 86, Dec. 1989, pp. 9558-9562.
Hammerschmidt et al., “Identification and Characterization of oriLyt, a Lytic Origin of DNA Repliction of Epstein-Barr Virus”, Cell, vol. 55, Nov. 4, 1988, pp. 427-433.
Hammerschmidt et al., “Genetic Analysis of Immortalizing functions of Epstein-Barr Virus in Human B Lympohocytes”,Nature, vol. 340, Aug. 3, 1989, pp. 393-397.
Hanahan “Studies of Transformation ofEscherichi coliwith Plasmids”,J. Mol. Biol., (1983) vol. 166, pp. 557-580.
Yates et al., “Stable Replication of Plasmids Derived From Epstein-Barr Virus in Various Mammalian Cells”,Nature, vol. 313, Feb. 28, 1985, pp. 812-815.
Firth et al., “Structure and Function of the F Factor and Mechanism of Conjugation”,Escherchia coli and Salmonella, Second Edition, vol. 2., 1996) pp. 2377-2401.
Site-specific insertion DNA into a pseudorabies virus vector, Sauer et al., Proc. Natl. Acad. Sci. USA, vol. 84, pp. 9108-9112, Dec. 1987 Genetics.
Delecluse Henri-Jacques
Hammerschmidt Wolfgang
Pich Dagmar
GSF Forshungszentrum fur Umwelt und Gesundheit GmbH Ingolstradte
Hunton & Williams
Wortman Donna C.
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