Expression of an exogenous gene in a mammalian cell by use of a non-mammalian DNA virus having an altered coat protein

#This invention relates to the use of a non-mammalian DNA virus to express an exogenous gene in a mammalian cell.

#Current methods for expressing an exogenous gene in a mammalian cell include the use of mammalian viral vectors, such as those that are derived from retroviruses, adenoviruses, herpes viruses, vaccinia viruses, polio viruses, or adeno-associated viruses. Other methods of expressing an exogenous gene in a mammalian cell include direct injection of DNA, the use of ligand-DNA conjugates, the use of adenovirus-ligand-DNA conjugates, calcium phosphate precipitation, and methods that utilize a liposome- or polycation-DNA complex. In some cases, the liposome- or polycation-DNA complex is able to target the exogenous gene to a specific type of tissue, such as liver tissue.

#Typically, viruses that are used to express desired genes are constructed by removing unwanted characteristics from a virus that is known to infect, and replicate in, a mammalian cell. For example, the genes encoding viral structural proteins and proteins involved in viral replication often are removed to create a defective virus, and a therapeutic gene is then added. This principle has been used to create gene therapy vectors from many types of animal viruses such as retroviruses, adenoviruses, and herpes viruses. This method has also been applied to Sindbis virus, an RNA virus that normally infects mosquitoes but which can replicate in humans, causing a rash and an arthritis syndrome.

#FIG. 1 is a schematic representation of the AcMNPV RSV-lacZ transfer plasmid pZ4.

#FIG. 2 is a schematic representation of the occluded AcMNPV RSV-lacZ transfer plasmid pZ5.

#FIG. 3 is a schematic representation of the episomal transfer plasmid pZ-EBV#1, a chimera of baculovirus and Epstein Barr Virus sequences. A virus produced with this transfer plasmid is capable of replicating in a mammalian cell.

#Genetic Manipulation of Viruses

#In contrast to conventional gene expression methods, the invention involves modifying non-mammalian DNA viruses that do not naturally infect and replicate in mammalian cells. Thus, the invention is based on the addition of new properties to a non-mammalian DNA virus that allow it to deliver a gene to a mammalian cell and direct gene expression within the mammalian cell. In contrast, conventional gene therapy vectors require that one disable viral functions, such as expression of viral genes and viral genome replication.

#In the present method, the viral particle serves as a “shell” for the delivery of DNA to the mammalian cell. The viral DNA is engineered to contain transcriptional control sequences that are active in a mammalian cell, to allow expression of the gene of interest in the target cell. Conventional recombinant DNA techniques can be used for inserting such sequences. Because the non-mammalian DNA viruses used in the invention are not capable of replicating in mammalian cells, it is not necessary to delete essential viral functions to render them defective. It is preferred, however, that the virus naturally replicate in a eukaryotic species (e.g., an insect, a plant, or a fungus). Examples of viruses that can be engineered to express an exogenous gene in accordance with the invention are listed in Table 1. Preferably, the genome of the virus used in the invention is normally transported to the nucleus in its natural host species because nuclear localization signals function similarly in invertebrate and in mammalian cells. The data summarized below show that, in contrast to conventional wisdom, a non-mammalian DNA virus can (1) infect a wide variety of mammalian cells, and (2) direct expression of an exogenous gene in the cells. In addition, expression of an altered coat protein on the surface of a virus particle enhances the ability of the virus to express an exogenous gene in a mammalian cell.