Boyce Thompson Institute for Plant Research

University of Iowa Research Foundation

Gary W Blissard

Scott A Monsma

Jodie T Mangor

Robert R Granados

Patrick Sinn

Paul Mccray

Guangyun Lin

Jian Zhou

Scott C Monsma

The present invention provides a method for restoring budding capability to GP64null baculoviruses including gp64null AcMNPV by expressing therein a portion of the VSV G protein gene or a truncated 'stem' portion of the GP64 gene. Other embodiments provide methods to use portions of the G-stem or GP64 protein to target foreign proteins for display on virions.

Baculoviruses with enhanced virion production and a method for the production of baculoviruses

A method of co-expressing a portion of the VSV G protein gene or a truncated 'stem' portion with GP64 and a retrovirus increases the titer of retroviral vectors. A truncated VSV G protein, preferably comprised of a small segment from the C-terminal portion of the ectodomain plus the transmembrane (TM) and cytoplasmic tail (CTD) domains of VSV G, co-expressed with retroviral vectors, enhances the production titers of the retroviral vectors. A preferred embodiment uses a VSV G construct that includes an N-terminal c-Myc epitope plus 42 amino acids from the C-terminal portion of the ectodomain, 20 amino acids from the predicted TM domain, and 29 amino acids from the predicted CTD of the VSV G protein.

Cell lines that are commonly used for protein expression are engineered to include genes that encode suppressors of apoptosis (SA). Insect cell lines expressing these SA genes are resistant to apoptosis or programmed cell death, and express certain recombinant proteins at increased levels. These cell lines also have increased resistance to many types of stress. Because some of the SA proteins inhibit apoptosis in a wide spectrum of organisms, these genes may be inserted into other plant or animal cell lines for a variety of purposes involving resistance to apoptosis or resistance to stress.

Stable cell lines resistant to apoptosis and nutrient stress and methods of making same

A pseudotyped baculovirus comprises a deletion, inactivation or reduction from regulation of a baculovirus envelope protein gene, and is engineered to express an envelope protein from another virus or cell, or another protein or molecule that facilitates entry of the baculovirus into a non-host cell, or provided with a heterologous envelope protein or another protein or molecule that facilitates entry of the baculovirus into a non-host cell by other suitable means. Such baculoviruses can be used to efficiently deliver genes to mammalian cells or organisms, and such genes can be expressed either from the baculovirus genome, or integrated into the mammalian cell genome, and can be used for expression of proteins such that purification of secreted or other protein products does not require removal of contaminating baculovirus particles or baculovirus envelope proteins.

GP64-null baculoviruses pseudotyped with heterologous envelope proteins for gene therapy

A pseudotyped baculovirus comprises a deletion, inactivation or reduction from regulation of a baculovirus envelope protein gene, and is engineered to express an envelope protein from another virus or cell, or another protein or molecule that facilitates entry of said baculovirus into a non-host cell, or provided with a heterologous envelope protein or another protein or molecule that facilitates entry of said baculovirus into a non-host cell by other suitable means. Such baculoviruses can be used to efficiently deliver genes to mammalian cells or organisms, and such genes can be expressed either from the baculovirus genome, or integrated into the mammalian cell genome, and can be used for expression of proteins such that purification of secreted or other protein products does not require removal of contaminating baculovirus particles or baculovirus envelope proteins.

GP64-null baculoviruses pseudotyped with heterologous envelope proteins

Disclosed herein is a novel baculovirus cloning system. The new cloning system is a marker-rescue system, using an essential gene, e.g. gp64. In this system, a gene essential for viral replication, growth, or propagation in cell culture is removed from or inactivated in the viral genome. Once a null baculovirus is created, it is propagated in a host cell that expresses the essential protein or a functional homolog. For cloning into the baculovirus containing the null-mutation, the virus is used to infect wild type host cells and the same cells are transfected with a plasmid that contains the essential gene, or a functional homolog, linked to a foreign gene under the control of a selected promoter. The baculovirus is 'rescued' by the rescue gene linked to the foreign gene and is able to propagate normally and express the foreign gene. The recombinant 'rescued' baculovirus can be used for gene expression, biological control or presentation of a foreign protein on the surface of the virus for vaccines and antibody production. As an example of this new cloning system, disclosed herein are recombinant baculoviruses that contain an insertionally inactivated or deleted gp64 efp gene, a gene that encodes a protein essential for viral infectivity and propagation in cell culture and in animals. To generate the virus the GP64 EFP protein was supplied in trans, from a stably transfected cell line. Homologous recombination was the used to generate inactivated gp64 efp genes in the context of otherwise wild type AcMNPV baculoviruses.

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Ithaca, NY, United States of America

Gary W Blissard