HSV viral vector

Details HSV viral vector HSV viral vector

1997-01-28

2003-09-02

Housel, James (Department: 1648)

Organic compounds -- part of the class 532-570 series

Organic compounds

Carbohydrates or derivatives

C424S199100, C424S231100, C435S069300, C435S235100, C435S320100, C514S04400A, C530S350000, C536S023100, C536S023500

Reexamination Certificate

active

06613892

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates to a recombinant herpes simplex virus (HSV), especially type 1 (HSV-1) or type 2 (HSV-2) having a good ability to continuously express an inserted heterologous gene whilst the virus is at the same time maintained in its latent non-replicative state.
BACKGROUND OF THE INVENTION
A distinguishing feature of herpes virus infections is the ability to persist in the host for long periods in a non-replicative or latent state. Herpes simplex virus type 1 (HSV-1) establishes latent infection in human peripheral sensory ganglia and can reactivate to produce recurrent mucocutaneous lesions. Operationally, the pathogenesis of herpes virus infections can be divided into several distinct stages which can be studied individually in experimental animal models: acute viral replication, establishment of latency, maintenance, and reactivation. Following inoculation, HSV-1 replicates at the site of inoculation and is transported to sensory ganglia. Replication at the periphery or in sensory ganglia may increase the amount of virus that can establish latent infection. During latent infection, HSV-1 DNA can be detected in infected tissues but infectious virus cannot be detected. This latent state is often maintained for the life of the host. A variety of stimulae (such as fibrile illness and exposure to ultraviolet irradiation) can interrupt the latent state and cause the reappearance of infectious virus or reactivation.
Transcription of the HSV-1 immediate early (IE) genes is not detectable during latency. However, in tissue culture, IE gene expression is a pre-requisite for viral replication. Transcription of the IE genes is transinduced by a virion protein Vmw65 (transinducing factor) that is a component of the HSV-1 virion. Vmw65 does not bind directly to HSV-1 DNA but mediates transinduction by association with cellular proteins to form a complex which interacts with the IE regulatory element.
Ace et al (1989) report an HSV-1 mutant which contains a 12 bp insertion in the coding region of Vmw65 which is unable to transinduce IE gene expression, though the altered Vmw65 is incorporated into mature virions.
The inventor's previous patent specification WO91/02788 discloses a herpes simplex virus type 1 mutant which includes the mutation within the Vmw65 gene which removes the transinducing properties of the Vmw65 transactivator protein such that the virus remains in its latent state. In addition, a &bgr;-galactosidase marker gene under the control of the latency associated transcript (LAT) promoter is inserted into the thymidine kinase (TK) gene and expression of the heterologous gene during latency is observed.
It is an object of the present invention to provide an HSV viral vector having enhanced expression of the inserted heterologous gene during latency.
SUMMARY OF THE INVENTION
Generally speaking, the present invention is based on the discovery that enhanced long term expression during latency may be obtained by use of the IE1 gene enhancer of cytomegalovirus controlling the inserted heterologous gene.
Most specifically, the present invention provides a recombinant herpes simplex virus (HSV) viral vector genome which comprises;
(i) a DNA sequence change in the gene coding for Vmw65 protein such as to substantially remove transinducing properties thereof; and
(ii) an expressable heterologous gene inserted into a region of the HSV genome which is non-essential for culture of the virus, the gene being under the control of the immediate early 1 (IE1) gene enhancer of cytomegalovirus (CMV).
DETAILED DESCRIPTION OF THE INVENTION
The Vmw65 sequence change removes the transinducing properties thereof such that expression of HSV IE genes and therefore HSV viral replication in vivo, is substantially removed. The HSV vector is therefore constrained to remain in its latent state. The use of the IE1 CMV enhancer to control the inserted heterologous gene has been found to give excellent long term expression of the heterologous gene during latency. Experiments in mice using the inserted heterologous lacZ gene have showed continuous expression from the latent vector of up to five months. In contrast, use of other promoters such as HSV-1 Vmw110 and Vmw65, and the Moloney murine leukaemia virus enhancer have been found not to give long term expression during latency.
The structure of the human cytomegalovirus (HCMV) enhancer is discussed in Stinski and Roehr (1985). The IE1 enhancer is the promoter-regulatory region upstream of the major immediate early gene of human cytomegalovirus. This enhancer region upstream of the IE1 gene consists of a series of different repeat sequences distributed up to −509 bp from the site for the initiation of transcription. Within this enhancer are a set of inducing sequences. Certain of the sequences within the enhancer region are non-essential and do not effect the level of expression obtained, whilst other sequences promote downstream expression.
The CMV enhancer is generally that derived from human cytomegalovirus (HCMV) and the immediate early 1 (IE1) nomenclature applies particularly to that virus. However, the analogous enhancer from other types of CMV, such as mouse, rat, equine, simian, and guinea pig CMV, may also be employed.
The present invention primarily envisages the use of the entire CMV IE1 enhancer sequence. Indeed in a preferred embodiment of the invention a larger sequence extending to −730 bp and including the entire CMV IE1 enhancer was employed. However, it is clearly within the ambit of the skilled man to modify the naturally occurring enhancer sequence without departing from the general scope of the present invention. Thus, the present invention is concerned not only with the use of the entire CMV IE1 enhancer sequence but also with variations in that sequence, either by insertion, deletion or substitution such that the enhancer properties are not substantially affected.
Other promoter sequences, such as the LAT (latency associated transcript) promoter, may be included upstream of the inserted heterologous gene and HMCV enhancer, but these have not been found to offer any particular advantage according to the present invention.
The position and size of the DNA sequence change in the gene coding for Vmw65 protein is significant, since it is necessary to substantially remove the transinducing properties of the Vmw65 protein (and thereby prevent in vivo replication of the virus and consequent illness of the patient), whilst at the same time retaining the structural properties of the protein required to successfully assemble the complete virion when the virus is cultured. The viral vector of the present invention must be capable of replication under culture conditions so as to be able to produce sufficient quantities of the mutant virus for use, but at the same time the virus should be incapable of replication in vivo. Preferably, the DNA sequence change is achieved by a transition (purine to purine or pyrimidine to pyrimidine) or transversion (purine to pyrimidine or vice versa) alteration of 1-72 base pairs, an oligonucleotide insert of 3-72 base pairs or a deletion of 3-72 base pairs, at a position between amino acids 289 and 480 (especially 289 and 412) of the Vmw65 protein.
The recombinant HSV may be of type HSV-1 or HSV-2 or may be an intertype recombinant between HSV-1 and HSV-2 which comprises nucleotide sequences derived from both types. The recombinant HSV genome will generally be contained in a mutant HSV virus.
HSV has the ability to infect many tissue types and therefore in principle the viral vector of the present invention may be used as a vector directed against a wide variety of cell types. Latency in HSV infection tends to be established within neuronal cells, though it is possible that expressed gene products may translocate from their original point of production. The viral vector of the present invention is thus particularly useful for delivering expressable heterologous genes into neuronal cells. The genes may deliver a therapeutic effect or may deliver an antigenic

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