Drug – bio-affecting and body treating compositions – Antigen – epitope – or other immunospecific immunoeffector – Virus or component thereof
Reexamination Certificate
2001-02-20
2004-04-13
Housel, James (Department: 1648)
Drug, bio-affecting and body treating compositions
Antigen, epitope, or other immunospecific immunoeffector
Virus or component thereof
C424S199100, C424S202100, C435S235100, C435S320100
Reexamination Certificate
active
06719982
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to mutant herpes simplex viruses which have inactivating mutations rendering them non-pathogenic. It also relates to the use of such mutant herpes simplex viruses in gene therapy and in methods of assaying for gene function.
BACKGROUND TO THE INVENTION
Herpes simplex virus (HSV) has often been suggested as a suitable vector for the nervous system due to its neurotrophic lifestyle and its ability to remain in neurons for the lifetime of the cell. However wild type HSV is highly pathogenic and must, like most viral vectors, be disabled in some way. The pathogenic effects of HSV result from lytic infection with the virus and therefore the use of HSV as a vector requires the development of strains carrying mutations that disrupt the lytic cycle whilst allowing the establishment of asymptomatic latent infections.
HSV vectors have previously been produced and tested in vivo by the deletion of the essential immediate early (IE) gene ICP4 (Dobson et al., 1990 and Chiocca et al., 1990), which must be complemented for growth in culture. ICP4 is required for transcriptional activation of the viral early and late genes in lytic infection. Thus, a virus lacking this gene can readily infect cells but cannot grow lytically. Another essential gene is ICP27, whose gene product is highly cytotoxic probably due to its secondary role of preventing the splicing of pre-mRNAs in favour of translation from the mainly unspliced herpes RNAs. HSV strains have been produced with deletions in ICP27, either in ICP27 alone (for example Reef Hardy and Sandri-Goldin, 1994 and Rice and Knipe, 1990), or in combination with ICP4 (for example U.S. Pat. No. 5,658, 724).
Mutations have also been made in non-essential genes such as the IE gene ICP0, the IE gene ICP6, tyrosine kinase (TK), US5 or VMW65, all of which are required for full pathogenicity in vivo but are dispensable for growth in culture (reviewed by Coffin and Latchman, 1996). These types of mutation provide the added advantage that the deletion need not be complemented for growth in culture, which has been shown previously to occasionally result in reversion of the non-pathogenic phenotype to a wild-type phenotype by homologous recombination between the virus and the complementing sequences in the cell-line during growth. However in each of these cases, mutation of the non-essential gene does not completely prevent virus replication since high titre inoculation will overcome the block to replication in vivo.
ICP34.5, the so-called neurovirulence factor, is absolutely required for neurovirulence in vivo, but is unnecessary for growth in culture (Chou et al., 1990). Mutations in ICP34.5 provide a subtle mechanism by which HSV can be disabled. ICP34.5 is thought to prevent the usual host response to a productive infection in neurons, which results (in the absence of ICP34.5) in cell death and thus the limitation of the infection to initially infected cells. ICP34.5 is thought to override this response and allow full lytic replication to occur. Thus in the absence of ICP34.5, if a disabled virus were ever to re-establish a productive infection for whatever reason, the ICP34.5 mutation would ensure that the protective host response limited virus replication to a small number of cells.
It is, however, unlikely that viruses carrying a single defect will be considered safe enough for eventual human use. Added safety and the possibility of higher titre inoculation might be achieved by the inactivation of an essential IE gene providing an absolute block to replication (and which must thus be complemented in culture), together with inactivation of a non-essential gene.
SUMMARY OF THE INVENTION
We have now found that herpes simplex viruses carrying inactivating mutations in at least ICP4 and ICP34.5 (optionally with inactivating mutations in VMW65 and/or vhs) exhibit reduced levels of toxicity compared to virus strains carrying mutations in ICP34.5 alone, ICP34.5 together with VMW65, or ICP34.5 together with VMW65 and vhs. They are safer than viruses deleted for ICP4 alone. These highly mutated strains can however still be grown efficiently in culture, using an ICP4 complementing cell line, allowing preparation of stocks of the virus. Furthermore, these HSV strains of the invention have been shown to be suitable vectors for delivery of heterologous genes to mammalian cells.
Thus the present invention provides a herpes simplex virus (HSV) having a functional ICP27 gene and which lacks at least a functional ICP4 gene and a functional ICP34.5 gene. Preferably said virus further lacks one or more functional non-essential genes other than the said ICP34.5 gene. For example the virus may further lack one or more functional non-essential genes selected from VMW65, vhs, ICP0, ICP6 and TK. Two or even three of these non-essential genes may thus be inactivated.
In particular, to reduce toxicity in vivo, we have inserted an inactivating mutation in the non-essential gene VMW65 of an HSV of the invention to reduce immediate-early (IE) gene expression, and thus also the level of expression of the genes regulated by these proteins. Furthermore, we have also produced herpes simplex viruses with deletions in the gene encoding the virion host shut-off protein (vhs). Vhs is a protein carried in the virion and is responsible for destabilising mRNA and thus reducing host protein synthesis in favour of translation from the more rapidly produced viral RNA which accompanies infection with a wild type virus.
Consequently, in a preferred embodiment of the present invention, the HSV of the invention also lacks a functional vhs gene and/or a functional VMW65 gene (due to a mutation in said VMW65 gene which abolishes its transcriptional-activation activity). A particularly preferred virus of the invention lacks a functional ICP4 gene, a functional ICP34.5 gene, a functional vhs gene and a functional VMW65 gene due to a mutation in said VMW65 gene which abolishes its transcriptional-activation activity.
The herpes simplex viruses of the invention can be used, for example, for delivering therapeutic genes in methods of treatment of diseases of, or injuries to, the nervous system, including Parkinson's disease, spinal injury or strokes, or diseases of the eye, heart or skeletal muscles, or malignancies. The present invention also relates to methods for studying the function of genes in mammalian cells, for example in identifying genes complementing cellular dysfunctions, or studying the effect of expressing mutant genes in wild-type or mutant mammalian cells. The methods of the present invention may be used in particular for the functional study of genes implicated in disease.
The invention further provides an HSV of the invention which carries a heterologous gene. The term heterologous gene is intended to embrace any gene not found in the HSV genome. The heterologous gene may be any allelic variant of a wildtype gene, or it may be a mutant gene. Heterologous genes are preferably operably linked to a control sequence permitting expression of said heterologous gene in mammalian cells, preferably cells of the central or peripheral nervous system, or cells of the eye, heart or skeletal muscle, more preferably cells of the central or peripheral nervous system. The HSV of the invention may thus be used to deliver a heterologous gene to a mammalian cell where it will be expressed. Such vectors are useful in a variety of applications, for example, in gene therapy, or in vitro assay methods or for the study of HSV gene regulation.
The heterologous gene preferably encodes a polypeptide of therapeutic use, including polypeptides that are cytotoxic or capable of converting a precursor prodrug into a cytotoxic compound.
The invention further provides herpes simplex viruses of the invention, carrying a heterologous gene, for use in the treatment of humans and animals. For example, such viruses may be used in the treatment of diseases of, or injury to, the nervous system, including Parkinson's disease, spinal injury or strokes or disease of the
Coffin Robert S.
Finnie Nicholas J.
Latchman David S.
Biovex Limited
Foley Shanon
Housel James
Nixon & Vanderhye P.C.
LandOfFree
Mutant herpes simplex viruses and uses thereof does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Mutant herpes simplex viruses and uses thereof, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Mutant herpes simplex viruses and uses thereof will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-3245854