Methods of expressing proteins in insect cells and methods...

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

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C435S348000, C435S455000, C435S456000, C435S320100

Reexamination Certificate

active

06221632

ABSTRACT:

BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to a method of expressing heterologous proteins in insect cells using genetic elements which potentiate activity of an insect cellular promoter functionally attached to a structural gene for a heterologous protein. The heterologous protein may include proteins toxic or harmful to an insect host or proteins whose unregulated expression will incapacitate the insect host. The invention is also directed to expression cassettes, recombinant expression cassettes containing heterologous genes, transplacement fragments containing expression cassettes, transplacement fragments containing recombinant expression cassettes, vectors containing tansplacement fragments and recombinant baculoviruses derived therefrom.
2. State of the Art
Nuclear polyhedrosis viruses (NPVs) are a subgroup of the family Baculoviridae, whose virions are embedded into proteinaceous polyhedra in the nucleus of host cells. Baculoviruses provide alternatives to chemicals for controlling insect pests. Because most NPVs have a host range restricted to only a few closely related species, they can be used without disrupting the balance of other insect and non-insect species (e.g. important predators) in the agricultural ecosystem. No baculovirus has been demonstrated to infect mammals, reptiles, birds, invertebrates such as earthworms or plants. To date, however, baculoviruses have met with only limited commercial success as control agents, due to difficulties with virus stability and, most importantly, slower speed of action than that achieved with chemical insecticides.
Certain baculoviruses, specifically nuclear polyhedrosis viruses (NPVs), have a unique life cycle which involves the temporally regulated expression of two functionally and morphologically different viral forms, the budded form and the occluded form. Nuclear polyhedrosis viruses produce large polyhedral occlusion bodies, which contain enveloped virus particles, within the nucleus of infected cells. The occlusion body is composed of a matrix comprising a 29 kDa protein known as polyhedron. After the insect dies from infection, occlusion bodies containing virus are released from the dead larvae into the environment and spread the infection to other insects through contamination of the food supply. These occlusion bodies serve to protect the virus particles in the environment and also provide a means of delivering the virus particles to the primary site of infection in insects, the midgut epithelial cells. When the occlusion bodies are ingested by the larvae, the alkaline pH of the midgut lumen of phytophagous lepidopteran larvae dissolves the paracrystalline matrix in which the virus particles are embedded, promoting infection.
Secondary infection within the insect involves the budded form of the virus. Viral nucleocapsids are synthesized in the nucleus of the insect cell, move through the cytoplasm and bud from the plasma membrane of the cell resulting in the release of budded virus particles into the insect hemolymph. The open circulatory system of the insect provides the virus with access to other tissues of the insect. Virtually all tissues within the host larvae are susceptible to infection by the budded virus. Replication of the virus in other organs creates extensive tissue damage and eventually death. Generally, the complete process can take 4-5 days in the laboratory, but may take more than a week in the field.
The synthesis of the budded and occluded forms of the virus is temporally regulated. During a typical infection of host tissue culture cells, progeny budded viruses are released into the culture media beginning approximately 12 hour post infection (p.i.) and the release continues logarithmically through 22 hours p.i. Occluded virus forms approximately at 20 hours p.i. and continues through 70 hours p.i. by which time approximately 70-100 polyhedral occlusions have formed in the nucleus. This temporal regulation of viral development is reflected in the controlled transcription of specific viral genes.
Nuclear polyhedrosis virus genes are transcribed in a regulated cascade involving at least three phases of transcription: an early phase (0-6 hours p.i.) prior to viral DNA replication, a late phase (6-18 hours p.i.) involving DNA replication and budded virus formation and the very late occlusion phase (18 through 70 hours p.i.).
In contrast to chemical pesticides which usually act upon the target insect immediately upon application, baculovirus infection-mediated reduction of the population of insects occurs in the field only one to two weeks after application of wild-type baculovirus. In order to increase the speed of insect inactivation by baculoviruses, recombinant viruses have been generated which express non-viral proteins whose products are toxic to the infected insect, under the control of viral or synthetic promoters. The underlying premise for the creation of such recombinant viruses has been that the expression of the foreign protein in the larvae should inactivate or kill the larvae before they would normally succumb to viral infection. Some recombinant viruses have been developed which employ the viral polyhedron promoter (Merryweather et al., 1990; Tomalski and Miller, 1991; Maeda et al., 1991), the viral p10 promoter (Stewart et al., 1991; McCutchen et al., 1991), or a synthetic promoter based on the previous two (Wang et al., 1991) to express the desired foreign protein. While these viral promoters can direct the expression of high levels of protein, they are not expressed until the very late occlusion stage of infection.
One of the key aspects of the development of recombinant baculoviruses as effective insecticides is the timing and site of expression of heterologous proteins following initial infection of the target insect. When larvae are infected orally with relatively low doses of polyhedra, as would normally occur under field conditions, the first cells to be infected are the columnar and regenerative cells of the midgut epithelium. The generalized spread of the virus to other tissues of an infected larva through circulation does not occur until 36 hours after the virus is first observed in the gut epithelium. Expression of heterologous genes under the control of the polyhedron or p10 promoters in vivo may not occur until an even later time as there is some doubt as to the level of expression of genes under the control of the viral polyhedron or p10 promoters in the epithelial cells of the midgut, the primary site of infection, as normal production of polyhedra is not observed in these cells (Granados and Lawler, 1981). Thus, placing an insect incapacitating or toxic gene under the control of the polyhedron or p10 promoter may offer modest advantages in the order of only 1 or 2 days in terms of accelerating insect death relative to an infection with a wild type baculovirus.
Because early viral promoters are usually essential and cannot be deleted, their utilization as the promoter for the toxin gene would require the presence of a duplication of the promoter sequence in the viral genome. Recombinant viruses containing such duplications of early viral promoters may prove unstable over the many large-scale passages necessary for commercial production.
A recombinant virus has also been developed which contains silkmoth chorion chromosomal genes under the control of their own promoter (Iatrou and Meidinger, 1990). The transcripts from this recombinant virus are expressed correctly only in the tissue in which this promoter is normally active, ovarian follicular cells of the insect, but not in any other tissues, for example fat body, other tissues of the abdomen such as muscles or ganglia or in non-expressing tissue culture cells such as Bm5 cells. Further, because of the presence of a thick basement membrane that completely surrounds each follicle, the later recombinant virus infects the follicle cells only in a limited fashion and only after a considerable time lag (e.g., 36-48 hours) after in vitro inoculation (injection) of the insect wit

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