United States Patent:  6,106,825

Assignee:  University of Florida (Gainesville, FL)

Filed:  May 7, 1997

This invention provides recombinant entomopoxvirus as a novel vector and method of using such vector for delivery and expression of genes of biological significance to vertebrate cells, including human cells. This invention includes the use of an early entomopoxvirus or like gene promoter to drive the expression of an heterologous gene product in a vertebrate cell infected with the recombinant entomopoxvirus vector. We have discovered that, while the entomopoxvirus vector of this invention does not replicate in the vertebrate cell, it enters vertebrate cell cytoplasm and achieves expression of an heterologous gene inserted into the rEPV without causing cytopathic or cytotoxic effects. We further provide a method for expression of the heterologous gene under control of late, strong entomopoxvirus gene promoters, vertebrate gene or viral promoters, and for stable integration of the heterologous gene delivered by the rEPV into the vertebrate cell.

BRIEF SUMMARY OF THE INVENTION

This invention provides novel vectors and a method of using such vectors comprising recombinant entomopoxviruses for delivery and expression of genes of biological significance to vertebrates, including humans. This invention includes the use of an early entomopoxvirus or like gene promoter to drive the expression of an heterologous gene product in a recombinant entomopoxvirus (rEPV) vector. We have discovered that, while the entomopoxvirus vector of this invention is apparently able to enter into the vertebrate cell cytoplasm and achieve expression of the heterologous gene, the virus itself does not appear to be able to replicate in the cell and, perhaps most importantly, does not appear to be cytotoxic or lead to cell death. Baculovirus, which has been used as a live recombinant vector for delivery of genes into vertebrate cells, depends on the cellular machinery for transcription. In addition, baculovirus enters the nucleus of the cell, raising concerns about the possibility of chromosomal integration and disruption of normal genomic expression.

We demonstrate herein that heterologous genes under the control of EPV early gene promoters, or promoters which are recognized by the EPV transcriptional factors present in the EPV viral particle and therefore behave like early EPV gene promoters, or promoters recognized by the vertebrate cell's transcriptional apparatus when the rEPV is directed to enter the vertebrate cell nucleus, are expressed when vertebrate cells are contacted with the rEPV, even though EPV does not productively infect vertebrate cells (i.e., fails to replicate and fails to express most of the entomopoxvirus specific proteins). EPV genes under control of late promoters are not expressed. The infected cells survive, and the protein expression is limited by the stability of the EPV-transcriptional apparatus, which remains in the cellular cytoplasm.

We prepared a series of AmEPV-lacZ recombinants wherein the lacZ gene is regulated by either late (the AmEPV spheroidin or cowpoxvirus A-type inclusion body (ATI)) promoters or early promoters (the AmEPV early strong promoter, esp, or Melolontha melolontha MmEPV fusolin viral promoters). When the recombinants were used to infect vertebrate cells, .beta.-galactosidase expression occurred (>30% cells) when the lacZ was regulated by either the fusolin or esp promoters but not where lacZ was regulated by the late spheroidin or ATI promoters. Therefore, we have found that recombinant EPV enters vertebrate cells, undergoes at least partial uncoating and early, but not late, viral genes are expressed. When rEPV encoding the jellyfish green fluorescent protein (gfp) under control of the esp promoter or the fusolin promoter was used to infect vertebrate cells, single fluorescent cells were observed which continued to divide over a period of several days, ultimately forming fluorescent cell clusters, demonstrating that vertebrate cells survive infection and continue to grow (see FIG. 6). Therefore, based on these findings, we conclude that entomopoxviruses may be used as efficient, non-toxic vectors for gene delivery to vertebrate cells for transient expression.

We demonstrate that rEPV encoding the .beta.-gal gene driven by an early entomopoxvirus promoter is capable of driving expression of .beta.-galactosidase upon infection of a vertebrate cell line in vitro. FIG. 1 shows the expression of this enzyme and evidence that the entomopoxvirus is not cytotoxic. This benign character of the virus is of tremendous value as it can be envisioned that, in vivo, the host organism will not be required to mount an immune response against this virus or cells in which it becomes housed. The key here is that only the early class of viral proteins are synthesized. As the late viral proteins are not efficiently synthesized in rEPV-infected vertebrate cells, the level of viral structural proteins is minimal or non-existent in this system. Hence this system provides for very low levels of foreign antigens against which the vertebrate immune system is required to react.

As a result of these features, one object of this invention is to provide an entomopoxvirus vector for use as a vertebrate gene delivery vehicle.

Other objects include:

Provision of a non-replicating vector for cytoplasmic expression of heterologous genes in vertebrate cells;

Establishment of a method for use of entomopoxviruses to deliver genes for transient or stable expression in vertebrates;

Establishment of methods for in situ expression of foreign genes when rEPV vectors are introduced into the lung, liver or other organ;

Construction of high-level entomopoxvirus vertebrate expression vectors by insertion of an inducible bacteriophage T7/lacZ cassette or analogous expression cassettes into the entomopoxvirus genome.

Provision of a novel vaccine vector wherein a recombinant EPV vector is used to deliver an effective amount of an immuno-stimulatory antigen.

Claim 1 of 25 Claims

1. A recombinant entomopoxvirus(rEPV) comprising an early entomopoxvirus gene promoter, an early vaccinia virus gene promoter or an early poxvirus promoter recognized by vertebrate transcriptional factors, wherein said promoter is operatively linked to a heterologous gene, and further comprising a nuclear localization signal directing translocation of said rEPV into a vertebrate cell nucleus.

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