Chemistry: molecular biology and microbiology – Animal cell – per se ; composition thereof; process of...
Reexamination Certificate
2000-03-30
2003-07-29
Nguyen, Dave T. (Department: 1632)
Chemistry: molecular biology and microbiology
Animal cell, per se ; composition thereof; process of...
C435S069100, C435S091400, C435S235100, C435S320100
Reexamination Certificate
active
06599739
ABSTRACT:
FIELD OF THE INVENTION
The field of the invention is related to infectious papillomavirus pseudoviral particles useful in gene transfer.
BACKGROUND OF THE INVENTION
Gene transfer is a laboratory strategy in which the genetic repertoire of eukaryotic cells is modified. Essentially, gene transfer involves the delivery, to target cells, of an expression cassette made up of one or more genes and the sequences controlling their expression. The transfer process is accomplished by delivery of the cassette to the cell where it can function appropriately.
Considerable effort has been made to develop delivery systems to express foreign proteins in eukaryotic cells. These systems can be divided into two types: transfection and infection.
The first type of delivery system for introducing cloned DNAs into eukaryotic cells involves transfection. Calcium phosphate- or DEAE-dextran-mediated transfection is the most widely used method. The polycation Polybrene allows the efficient and stable introduction of plasmid DNAs into cultured cells that are relatively resistant to transfection by other methods (Kawai, S., and Nishizawa, M., 1984, Mol. Cell. Biol. 4, 1172; Chaney, W. G., et al., 1986, Somatic Cell Mol. Genet. 12, 237). In protoplast fusion, protoplasts derived from bacteria carrying high numbers of copies of a plasmid of interest are mixed directly with cultured mammalian cells, and fusion of the cell membranes is accomplished with polyethylene glycol, with the result that the contents of the bacteria are delivered into the mammalian cells (Schaffner, W., 1980, Proc. Natl. Acad. Sci. USA 77, 2163; Rassoulzadegan, M., et al. 1982, Nature 295, 257). Electroporation features the application of electric pulses to mammalian and plant cells so that DNA is taken directly into the cell cytoplasm (Neumann, E., et al., 1982, EMBO J. 1, 841; Zimmermann, U., 1982, Biochim. Biophys. Acta 694, 227). Artificial membrane vesicles, such as liposomes and cationic lipids, are useful as delivery vehicles in vitro and in vivo (Mannino, R. J., and Gould-Fogerite, S., 1988, BioTechniques 6, 682; Felgner, P. L., and Holm, M., 1989, Bethesda Res. Lab. Focus 11, 21; Maurer, R. A., 1989, Bethesda Res. Lab. Focus 11, 25). Direct microinjection into nuclei is effective, but it cannot be used to introduce DNA on a large scale (Capecchi, M. R., 1980, Cell 22, 479). Finally, naked DNA can, by itself, be placed into cells by particle bombardment (Yang, N. S., et al., 1990, Proc. Natl. Acad. Sci. USA 87, 9568), or taken up by cells, particularly when injected into muscle (Wolff, J. A., et al., 1990, Science
247, 14651).
The other type of delivery system is mediated by infection and involves the use of viral expression vectors derived from simian virus 40 (SV40) (Elder, J. T., et al., 1981, Annu. Rev. Genet. 15, 295; Gething, M. J., and Sambrook, J., 1981, Nature 293, 620; Rigby, P. W. J., 1982,
Genetic engineering
, R. Williamson, ed., Academic Press, London, vol. 3, p. 83; Rigby, P. W. J., 1983, J. Gen. Virol. 64, 255; Doyle, C., et al., 1985, J. Cell. Biol. 100, 704; Sambrook, J., et al., 1986, Mol. Biol. Med. 3, 459), vaccinia virus (Mackett, M., et al., 1985,
DNA cloning: A practical approach
, D. M. Glover, ed., IRL Press, Oxford, vol. 2, p. 191; Moss, B., 1985,
Virology
, B. N. Fields, et al., eds., Raven Press, New York, p. 685; Fuerst, T. R., et al., 1986, Proc. Natl. Acad. Sci. USA, 83, 8122; Fuerst, T. R., et al., 1987, Mol. Cell. Biol. 7, 2538), adenovirus (Solnick, D., 1981, Cell 24, 135; Thummel, C., et al., 1981, Cell 23, 825; Thummel, C., et al., 1982, J. Mol. Appl. Genet. 1, 435; Thummel, C, et al., 1983, Cell 33, 455; Mansour, S. L., et al., 1985, Proc. Natl. Acad. Sci. USA 82, 1359; Karlsson, S., et al., 1986, EMBO J. 5, 2377; Berkner, K. L., 1988, BioTechniques 6, 616), retroviruses (Dick, J. E., et al., 1986, Trends Genet. 2, 165; Gilboa, E., et al., 1986, BioTechniques 4, 504; Eglitis, M. A., and Anderson, W. F., 1988, BioTechniques 6, 608), and baculoviruuses (Luckow, V. A., and Summers, M. D., 1988, Bio/Technology 6, 47).
Expression of proteins from cloned genes in eukaryotic cells has been used for a number of different purposes: to confirm the identity of a cloned gene by using immunological or functional assays to detect the encoded protein, to express genes encoding proteins that require posttranslational modifications such as glycosylation or proteolytic processing, to produce large amounts of proteins of biological interest that are normally available in only limited quantity from natural sources, to study the biosynthesis and intracellular transport of proteins following their expression in various cell types, to elucidate structure-function relationships by analyzing the properties of normal and mutant proteins, to express intron-containing genomic sequences that cannot be transcribed correctly into mRNA in prokaryotes, and to identify DNA sequence elements involved in gene expression. Because expression of proteins can serve so many different purposes, there is a need for new delivery systems to meet the challenge of getting foreign DNA into eukaryotic cells. The invention satisfies this need.
These and other objects of the invention will be apparent to one of ordinary skill in the art upon consideration of the specification as a whole.
SUMMARY OF THE INVENTION
In one aspect, the invention provides an infectious papillomavirus pseudoviral particle.
In another aspect, the invention provides a HPV16{BPV1} virion.
In still another aspect, the invention provides an infectious papillomavirus pseudoviral particle comprising: a papillomavirus vector DNA which comprises an E2 binding site and an expression cassette comprising a gene and a sequence controlling expression of the gene; and a papillomavirus capsid which comprises L1 and L2 structural proteins, such that the capsid encapsidates the vector DNA, where the gene is derived from a first biological species and the L1 structural protein is derived from a second biological species and the first biological species is different from the second biological species.
In yet another aspect, the invention provides the here-described infectious papillomavirus pseudoviral particle, where the first biological species is BPV1 and the second biological species is HPV16.
In a different embodiment, the invention relates to a method of making infectious papillomavirus pseudoviral particles comprising: providing a cell line which expresses papillomavirus E2 DNA binding protein and L1 and L2 structural proteins; transforming the cell line with a papillomavirus vector DNA which comprises an E2 binding site and an expression cassette comprising a gene and a sequence controlling expression of the gene, where the papillomavirus E2 binding site is a cognate binding site of the E2 DNA binding protein, and where the gene is derived from a first biological species and the L1 structural protein is derived from a second biological species and the first biological species is different from the second biological species; providing conditions for the encapsidation of the vector DNA by a capsid which comprises the L1 and L2 structural proteins to generate the particles; and harvesting the particles.
In the above method, the cell line may be a mammalian cell line, an insect cell line, or a yeast cell line.
In yet a different embodiment, the invention relates to a cell line comprising the here-described infectious papillomavirus pseudoviral particle.
In still a different embodiment, the invention relates to a method of transferring a gene into a cultured mammalian cell comprising: providing the here-described infectious papillomavirus pseudoviral particle; and infecting a cultured mammalian cell with the particle such that the cultured mammalian cell is transformed with the gene.
In another manifestation, the invention provides a method of screening for infectious papillomavirus pseudoviral particles comprising administering test particles to cultured mammalian cells capable of being infected thereby and scoring for infectivity thereof.
In a further manifestat
Lowy Douglas R.
Roden Richard B.
Schiller John T.
Knobbe Martens Olson & Bear LLP
Nguyen Dave T.
The United States of America as represented by the Department of
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