Chemistry: molecular biology and microbiology – Virus or bacteriophage – except for viral vector or...
Reexamination Certificate
2000-03-08
2004-12-14
Foley, Shanon (Department: 1648)
Chemistry: molecular biology and microbiology
Virus or bacteriophage, except for viral vector or...
C435S091420, C435S320100, C435S325000, C435S462000, C435S463000, C435S466000, C435S475000, C435S476000, C435S477000, C536S023720
Reexamination Certificate
active
06830920
ABSTRACT:
BACKGROUND OF THE INVENTION
Adenoviruses (Ad) are double-stranded linear DNA viruses with a 36 kb genome. Several features of adenovirus have made them useful as a transgene delivery vehicles for therapeutic applications, such as facilitating in vivo gene delivery. Recombinant adenovirus vectors have been shown to be capable of efficient in situ gene transfer to parenchymal cells of various organs, including the lung, brain, pancreas, gallbladder, and liver. This has allowed the use of these vectors in methods for treating inherited genetic diseases, such as cystic fibrosis, where vectors may be delivered to a target organ. In addition, the ability of the adenovirus vector to accomplish in situ tumor transduction has allowed the development of a variety of anticancer gene therapy methods for non-disseminated disease. In these methods, vector containment favors tumor cell-specific transduction.
Adenovirus vectors also are very important tools for deciphering the role of various proteins in biological processes in vitro and in vivo
1-4
. They are commonly used because they infect a wide variety of cell types, provide very high protein expression, and when purified show little prep-to-prep variation. The technology to generate the viruses requires only basic laboratory techniques. However, to progress from a cDNA of interest in a shuttle vector to a purified, wildtype-free virus traditionally involves many steps and requires a significant time investment.
Several approaches traditionally have been used to generate the recombinant adenoviruses. One approach involves direct ligation of restriction endonuclease fragments containing a transgene to portions of the adenoviral genome. The low efficiency of large fragment ligations and the scarcity of unique restriction sites, however, have made this approach technically challenging.
Alternatively, the transgene may be inserted into a defective adenovirus by homologous recombination results. The desired recombinants are identified by screening individual plaques generated in a lawn of complementation cells. Though this approach has proven useful, the low efficiency of homologous recombination, the need for repeated rounds of plaque purification, and the long times required for completion of the viral production process has hampered more widespread use of adenoviral vector technology.
Most adenovirus vectors are based on the adenovirus type 5 (Ad5) backbone in which an expression cassette containing the foreign gene has been introduced in place of the early region 1 (E1) or early region 3 (E3). Viruses in which E1 has been deleted are defective for replication and are propagated in human complementation cells (e.g., 293 or 911 cells), which supply the missing gene products provide the E1 and pIX products in trans.
Many laboratories continue to use standard methods of homologous recombination with shuttle plasmids and full-length Ad backbones (restricted in E1) for generation of vectors for basic research
5
. However, the time required to generate the vectors can range from a best-case scenario of 2 months to many months. Also, there may be wildtype contamination in the initial plaque isolation that necessitates further, time intensive, serial plaque isolations and amplification. Recent efforts have been directed at solving both the time and wildtype contamination problems. These include
E. coli
recombination methods
6
, ligation of cDNA directly into plasmids containing E1 deleted full-length viral DNA
7-9
, and an in vitro enzymatic recombination using Cre-loxP shuttles and backbone viral DNA
10-12
.
The advantages of these systems are that the repetitive plaque isolation to purify the viral particle can be avoided because there is no
6,8,12
or limited
10
wildtype viral DNA input. Thus, there is a reduction in the amount of time it takes to progress from the transfection of viral DNA to amplified, purified virus. However, these methods also have their drawbacks. For the
E. coli
recombination using plasmids containing adenovirus genomes, the system has high fidelity, but is inefficient and requires the screening of many bacterial colonies. This results in a significant time commitment even before transfection of recombinant DNA into E1-expressing cells such as HEK293 cells
13
. Similarly, ligation and recombinase methods require that several steps be completed before transfection into helper cell lines to generate virus. These recently developed methods are useful for making one or two viruses, but they are cumbersome if studies require multiple viruses to be generated.
Thus, to progress from a cDNA of interest in a shuttle vector to a purified, wild-type free virus is cumbersome as it involves many steps and can require a significant time investment. The time required to generate the vectors can range from a best-case scenario of two months to many months. Also, there may be wild-type contamination in the initial plaque isolation that necessitates further, time intensive, repetitions of plaque isolation and amplification.
Therefore, there is a continuing need for improved methods to accomplish multiple vector production in a simple and time-efficient manner.
SUMMARY OF THE INVENTION
The invention provides an Adenovirus (Ad) backbone plasmid comprising an Ad genome lacking map units 0 to 9.2, starting with a lefthand ITR. Further, any or all open reading frames constituting E4 or E3 may be modified in the Ad backbone plasmid. The modification may be a substitution, insertion, or deletion of one or more nucleotides, including being modified to contain a multiple cloning site. The Ad backbone plasmid may contain one or more genes required for Herpes Simplex Virus (HSV) packaging and/or an HSV origin of replication within the E3 region or other locations within the backbone. The plasmid may further comprise HSV Amplicon sequences required for packaging and replication, and the Amplicon sequences may be positioned on either side of the Ad left and right ITRs.
The invention further provides a shuttle plasmid comprising Ad sequences from 0 to 1 and 9.2 to 16.1 map units of an Ad genome. PacI restriction endonuclease sites may flank either end of the Ad sequences, and a multiple cloning site may be positioned between 1 and 9.2 map units. The shuttle plasmid may contain a sequence encoding a gene of interest, and may contain a novel promoter, inducible promoter or other sequence used to drive expression from a transgene.
The present invention also provides a cloning system for generating recombinant adenovirus comprising any of the Ad backbone plasmids described above and any of the shuttle plasmids described above.
The present invention further provides a host cell comprising any of the Ad backbone plasmids described above and any of the shuttle plasmids described above. The cell may express E1 sequences and pIX necessary for supporting adenovirus replication, and may express E4 sequences required for amplification of viruses generated with the modified Ad backbone. The cell may be an animal cell.
The present invention also provides method for rapidly producing recombinant adenovirus comprising contacting a host cell with any of the Ad backbone plasmids described above and any of the shuttle plasmids described above. This method may include the additional step of serially amplifying virus produced by the host cell and a step of detecting the presence of wild type virus. The shuttle plasmid used in the method may comprise a sequence encoding a gene of interest.
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patent: 5922576 (1999-07-01), He et al.
patent: 5965441 (1999-10-01), Breakefield et al.
patent: 6251677 (2001-06-01), Wilson et al.
patent: 6251957 (2001-06-01), Wilson et al.
patent: 0955373 (1999-11-01), None
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patent: WO 98/39035 (1998-11-01), None
Aoki et al. 1999. Efficient generation of recombinant adenoviral vectors by Cre-lox recombination in vitro. Molecular Medicine. vol. 5, pp. 224-231.*
Krougliak et al. 1995. Development of cell lines capable of complementing E1, E4, and pro
Anderson Richard
Davidson Beverly L.
Haskell Ronald
Xia Haibin
Fish & Richardson P.C. P.A.
Foley Shanon
University of Iowa Research Foundation
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