Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
1994-12-19
2001-07-24
Adams, Donald E. (Department: 1648)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S320100, C424S232100, C424S199100, C424S208100
Reexamination Certificate
active
06265183
ABSTRACT:
BACKGROUND OF THE INVENTION
The present invention relates to modified genomes of eukaryotic DNA viruses which replicate in the cytoplasm of a host cell, such as poxviruses and iridoviruses. More specifically, the invention relates to direct molecular cloning of a modified cytoplasmic DNA virus genome that is produced by modifying under extracellular conditions a purified DNA molecule comprising a cytoplasmic DNA virus genome. The modified DNA molecule is then packaged into infectious virions in a cell infected with a helper cytoplasmic DNA virus. In a preferred embodiment of the present invention, a foreign DNA fragment comprising a desired gene is inserted directly into a genomic poxvirus DNA at a restriction endonuclease cleavage site that is unique in the viral genome, and the modified viral DNA is packaged into virions by transfection into cells infected with a helper poxvirus.
Cytoplasmic DNA viruses of eukaryotes include diverse poxviruses and iridoviruses found in vertebrates and insects. Poxviruses having recombinant genomes have been used for expression of a variety of inserted genes. Such poxviruses can be used to produce biologically active polypeptides in cell cultures, for instance, and to deliver vaccine antigens directly to an animal or a human immune system. Construction of recombinant iridovirus genomes for expression of foreign genes appears not to be documented in the literature pertaining to genetic engineering.
Conventional techniques for construction of recombinant poxvirus genomes comprised of foreign genes rely in part on in vivo (intracellular) recombination. The use of intracellular recombination was first described as a process of “marker rescue” with subgenomic fragments of viral DNA by Sam and Dumbell,
Ann. Virol
. (Institut Pasteur) 132E:135 (1981). These authors demonstrated that a temperature-sensitive vaccinia virus mutant could be “rescued” by intracellular recombination with a subgenomic DNA fragment of a rabbit poxvirus. The methods they used for intracellular recombination are still used today.
Construction of recombinant vaccinia viruses comprised of non-poxvirus (“foreign”) genes was later described by Panicali and Paoletti,
Proc. Nat'l Acad. Sci. U.S.A.
79:4927-4931 (1982); Mackett et al.,
Proc. Nat'l Acad. Sci. U.S.A.
79:7415-7419 (1982); and U.S. Pat. No. 4,769,330. More specifically, the extant technology for producing recombinant poxviruses involves two steps. First, a DNA fragment is prepared that has regions of homology to the poxvirus genome surrounding a foreign gene. Alternatively, an “insertion” plasmid is constructed by in vitro (extracellular) ligation of a foreign gene with a plasmid. This plasmid comprises short viral DNA sequences that are homologous to the region of the poxvirus genome where gene insertion is ultimately desired. The foreign gene is inserted into the plasmid at a site flanked by the viral DNA sequences and, typically, downstream of a poxvirus promoter that will control transcription of the inserted gene. In the second step, the insertion plasmid is introduced into host cells infected with the target poxvirus. The gene is then indirectly inserted into the poxvirus genome by intracellular recombination between homologous viral sequences in the poxvirus genome and the portion of the plasmid including the foreign gene. The resulting recombinant genome then replicates, producing infectious poxvirus.
Thus, insertion of each particular gene into a poxvirus genome has heretofore required a distinct plasmid comprised of the gene flanked viral sequences selected for a desired insertion location. A difficulty with this approach is that a new insertion plasmid is required for each recombinant poxvirus. Each plasmid must be constructed by extracellular recombinant DNA methods, amplified in a bacterial cell, and then laboriously isolated and rigorously purified before addition to a poxvirus-infected host cell.
Another problem with extant methodology in this regard is a low yield of recombinant genomes, which can necessitate screening hundreds of individual viruses to find a single desired recombinant. The poor yield is a function of the low frequency of individual intracellular recombination events, compounded by the requirement for multiple events of this sort to achieve integration of the insertion plasmid into a viral genome. As a result, the majority of viral genomes produced by intracellular recombination methods are parental genomes that lack a foreign gene. It is often necessary, therefore, to introduce a selective marker gene into a poxvirus genome, along with any other desired sequence, to permit ready detection of the required rare recombinants without the need of characterizing isolated DNA's from numerous individual virus clones.
Purified DNA's of eukaryotic cytoplasmic DNA viruses are incapable of replicating when introduced into susceptible host cells using methods that initiate infections with viral DNA's that replicate in the nucleus. This lack of infectivity of DNA's of cytoplasmic DNA viruses results from the fact that viral transcription must be initiated in infected cells by a virus-specific RNA polymerase which is normally provided inside infecting virions.
“Reactivation” of poxvirus DNA, in which genomic DNA inside an inactivated, noninfectious poxvirus particle was packaged into infectious virions by coinfection with a viable helper poxvirus, has been known for decades. See, for instance, Fenner and Woodroofe,
Virology
11:185-201 (1960). In 1981 Sam and Dumbell demonstrated that isolated, noninfectious genomic DNA of a first poxvirus could be packaged into infectious poxvirus virions in cells infected with a second, genetically distinct poxvirus. Sam and Dumbell,
Ann. Virol
. (Institut Pasteur) 132E:135 (1981). This packaging of naked poxvirus DNA was first demonstrated by transfection of unmodified DNA comprising a first wildtype orthopoxvirus genome, isolated from virions or infected cells, into cells infected with a second naturally-occurring orthopoxvirus genome. However, heterologous packaging, packaging of DNA from one poxvirus genus (orthopox, for example) by viable virions of another genus (e.g., avipox), has not been demonstrated yet.
The use of intracellular recombination for constructing a recombinant poxvirus genome expressing non-poxvirus genes was reported shortly after Sam and Dumbell first reported intracellular packaging of naked poxvirus DNA into poxvirus virions and marker rescue with DNA fragments by intracellular recombination. See Panicali and Paoletti, 1982; Mackett et al., 1982. The relevant literature of the succeeding decade, however, appears not to document the direct molecular cloning, i.e., construction solely by extracellular genetic engineering, of a modified genome of any eukaryotic cytoplasmic DNA virus, particularly a poxvirus. The literature does not even evidence widespread recognition of any advantage possibly realized from such a direct cloning approach. To the contrary, an authoritative treatise has stated that direct molecular cloning is not practical in the context of genetic engineering of poxviruses because poxvirus DNA is not infectious. F. Fenner et al.,
THE POXVIRUSES
. Academic Press, 1989). Others working in the area have likewise discounted endonucleolytic cleavage and religation of poxvirus DNA's, even while recognizing a potential for rescue by infectious virus of isolated DNA comprising a recombinant poxvirus genome. See, for example, Mackett and Smith,
J. Gen. Virol.
67:2067-2082 (1986). Moreover, recent reviews propound the thesis that the only way feasible to construct a recombinant poxvirus genome is by methods requiring intracellular recombination. See Miner and Hruby,
TIBTECH
8:20-25 (1990), and Moss and Flexner,
Ann. Rev. Immunol.
5:305-324 (1987).
Vaccinia virus is a member of the Orthopox genus of the Poxvirus family with little virulence for humans. Although the exact origin of vaccinia virus is obscure, it is related to the cowpox virus used by Jenner and strains of vaccinia virus beca
Dorner Friedrich
Falkner Falko Gunter
Pfleiderer Michael
Scheiflinger Friedrich
Adams Donald E.
Baxter Aktiengesellschaft
Foley & Lardner
Parkin Jeffrey S.
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