Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound containing saccharide radical
Reexamination Certificate
1999-05-10
2001-09-18
Schwartzman, Robert A. (Department: 1636)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing compound containing saccharide radical
C435S320100, C435S325000, C435S252330, C435S254210, C536S023100, C536S024100
Reexamination Certificate
active
06291214
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention provides versatile vectors for efficient production of animal viral vectors carrying a nucleic acid of interest. Thus the vectors can be used to produce viral vectors for use in expression of nucleic acids, including production of RNA, antisense nucleic acids and polypeptides of interest.
2. Background
Adenoviruses (Ad) are 36 kb, linear, double-stranded DNA viruses that have been widely used for gene therapy, functional genetics, vaccines, and protein production (1-5). Most adenoviral vectors are based on the Ad type 5 (Ad5) viral backbone in which an expression cassette replaces the El and/or E3 region. Such E1
−
, E3
−
viruses are attractive vectors because they: i) can accept about 8 kb of exogenous DNA, ii) can be produced at very high titer in E1 complementing cells, and iii) are replication defective. Other attractive features of Ad vectors include infection of a wide range of both dividing and quiescent cell types at high efficiency, expression of genes at very high levels, and association with mild, self-limiting pathologies in humans (1-6). The increasing numbers of genes discovered by the human genome project, and the expanding field of gene therapy are driving a need for more efficient systems to generate recombinant Ad vectors to facilitate rapid, high throughput functional and therapeutic analysis.
Because of their complex genome, manipulation of Ad vectors to produce recombinant viruses has been difficult. Traditional methods of making recombinant adenoviruses use homologous recombination between two transfected DNAs in adenoviral-producing cell lines (6). The homologous recombination method is inefficient, time-consuming, and subject to contamination with replication-competent virus. Some newer methods of generating recombinant adenoviral vectors in yeast and
E. coli
have been reported (7-10). These methods shift the homologous recombination step from eukaryotic packaging cells into either yeast or
E. coli
. These newer methods allow the true cloning of the recombinant viral genome and thereby remove the need for clonal selection via repeated rounds of plaque purification. Although homologous recombination is quite efficient in yeast, it is desirable (because of the faster doubling time and ease of manipulation) to be able to do these steps in
E. coli
. However, homologous recombination in
E. coli
is not very efficient. Furthermore, homologous recombination with adenoviral sequences necessitates cloning each gene of interest into a vector that can only be used for adenoviral expression.
A method that has been used to construct recombinant baculoviruses in
E. coli
by Tn7-mediated site-specific transposition (11) has proven to be very efficient at generating recombinant baculoviruses. However, this system has several limitations.
The present invention overcomes many limitations on previous systems. Specifically, the present invention provides a Tn7-based transposition system for generating recombinant viruses, such as adenovirus, in
E. coli
. A low copy
E. coli
homing plasmid, containing a full length viral genome with lacZattTn7 inserted in a manner that does not interfere with later production of recombinant virus, has been constructed. The inventive system moves all sequences necessary for replication and selection in
E. coli
outside of the viral genome, allowing application to small animal viruses (Ad, AAV and retroviruses); there is no need for a selectable marker within the transposon allowing additional space for exogenous nucleic acid of interest; it allows construction of a variety of homing vectors for different viruses comprising different promoters such that one transfer vector can be used to express an exogenous DNA from numerous promoters in numerous viruses. Thus the system is very easy, rapid, and efficient, accommodates sizable DNA inserts, and generates truly clonal viruses. The homing vector system is easily adapted to allow subcloning into a single universal transfer vector that can be used to transpose genes into any of several different expression systems, thus facilitating cost-effective subcloning into a variety of vectors, including adenoviral, retroviral, adeno-associated viral, baculoviral and
E. coli
vectors.
SUMMARY OF THE INVENTION
The present invention provides an isolated nucleic acid, termed a homing vector, for generating a recombinant animal virus comprising
a) an animal virus polynucleotide wherein the viral polynucleotide comprises viral elements sufficient for recombinant viral production in a host cell upon contact with replication proteins of the virus;
b) a transposon target site within the virus polynucleotide wherein location of the transposon target site within the virus polynucleotide does not prevent recombinant viral production in a host cell upon contact with viral replication proteins; and
an origin of replication.
The present invention further provides transfer vector for transposition of an exogenous nucleic acid, comprising
(a) a transposon having a cloning site between a left end of the transposon and a right end of the transposon;
(b) a bacterial origin of replication positioned outside of a region encompassed by the left end and the right end of the transposon; and
a selectable marker.
The present invention further utilizes these two components in a system for generating recombinant animal viruses. Thus, the invention provides a kit as well as a method of producing a recombinant animal virus comprising
(a) contacting (1) a nucleic acid for generating a recombinant animal virus wherein the nucleic acid comprises
(i) an animal virus polynucleotide wherein the viral polynucleotide comprises viral elements sufficient for recombinant viral production in a host cell upon contact with replication proteins of the virus;
(ii) a transposon target site within the viral polynucleotide wherein location of the transposon target site within the viral polynucleotide does not prevent recombinant virus production in a host cell upon contact with viral replication proteins; and
(iii) an origin of replication,
in a cell under conditions suitable for transposition, with (2) a transfer vector, wherein the transfer vector comprises
(i) a transposon that recognizes the transposon target site,
(ii) an exogenous polynucleotide inserted between a left end of the transposon and a right end of the transposon,
(iii) a bacterial origin of replication positioned outside of a region encompassed by the left end and the right end of the transposon, and
(iv) a selectable marker,
to produce a transposition product; and
(b) transferring the transposition product into a cell comprising a viral replication protein, thereby producing the recombinant animal virus.
REFERENCES:
patent: 5348886 (1994-09-01), Lee et al.
patent: 5478731 (1995-12-01), Short
patent: 5846528 (1998-12-01), Podsakoff et al.
patent: 5851766 (1998-12-01), Ryals et al.
patent: 6004797 (1999-12-01), Colosi
Berkner, K. L., “Development of Adenovirus Vectors for the Expression of Heterologous Genes”, Bio Techniques, (1988) 6, 616-629.
Ketner, G. et al., “Efficient Manipulation of the human adenovirus genome as an infectious yeast artifical chromosome clone”, Proc. Natl. Acad. Sci USA 91 (1994) 6186-6190.
Chartier, C. et al., “Efficient Generation of Recombinant Adenovirus Vectors by Homologous Recombination inEscherichia coli”, J. Virol 70, (1996) 4805-4810.
Crouzet, J. et al., “Recombinational construction in Escherichia of infectiuos adenoviral genomes”, Proc. Natl. Acad. Sci. USA 94 (1997) 1414-1419.
He, T.-C, et al., “A Simplified system for generating recombinant adenoviruses”, Proc Natl. Acad. Sci USA 95 (1998) 2509-2514.
Luckow, V. A. et al., “Efficient Generation of Infectious Recombinant Baculoviruses by Site-Specific Transposon-Mediated Insertion of Foreign Genes into a Baculovirus Genome Propagated inEscherichia coli”, J. Virol 67, (1993) 4566-4579.
Richards Cynthia Ann
Weiner Michael Phillip
Davis Katharine F
Glaxo Wellcome Inc.
Schwartzman Robert A.
Selby Elizabeth
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