Combination of a fusogenic glycoprotein, pro-drug activation and oncolytic HSV as an intravesical therapy for superficial bladder cancer.

BACKGROUND: There are still no effective treatments for superficial bladder cancer (SBC)/non-muscle invasive bladder cancer. Following treatment, 20% of patients still develop metastatic disease. Superficial bladder cancer is often multifocal, has high recurrences after surgical resection and recurs after intravesical live Bacillus Calmette-Guérin. Oncovex(GALV/CD), an oncolytic herpes simplex virus-1, has shown enhanced local tumour control by combining oncolysis with the expression of a highly potent pro-drug activating gene and the fusogenic glycoprotein. METHODS: In vitro fusion/prodrug/apoptotic cell-based assays. In vivo orthotopic bladder tumour model, visualised by computed microtomography. RESULTS: Treatment of seven human bladder carcinoma cell lines with the virus resulted in tumour cell killing through oncolysis, pro-drug activation and glycoprotein fusion. Oncovex(GALV/CD) and mitomycin C showed a synergistic effect, whereas the co-administration with cisplatin or gemcitabine showed an antagonistic effect in vitro. Transitional cell cancer (TCC) cells follow an apoptotic cell death pathway after infection with Oncovex(GALV/CD) with or without 5-FC. In vivo results showed that intravesical treatment with Oncovex(GALV/CD) + prodrug (5-FC) reduced the average tumour volume by over 95% compared with controls. DISCUSSION: Our in vitro and in vivo results indicate that Oncovex(GALV/CD) can improve local tumour control within the bladder, and potentially alter its natural history.

Hepatocyte growth factor incorporated into herpes simplex virus vector accelerates facial nerve regeneration after crush injury.

Hepatocyte growth factor (HGF) promotes regeneration of the central nervous system, but its effects on the peripheral nervous system remain unclear. This study was conducted to elucidate the effect of HGF on regeneration of the murine facial nerve after crush injury. To do so, a replication-defective herpes simplex virus vector that incorporated HGF was prepared (HSV-HGF). The main trunk of the facial nerve was compressed by mosquito hemostats, and HSV-HGF, control vector or medium was then applied to the compressed nerve. We found that mice in the HGF group required significantly fewer days for complete recovery from nerve compression. Furthermore, the amplitude of the evoked buccinator muscle compound action potential increased following HSV-HGF application. HGF expression in and around the compressed nerve was demonstrated by enzyme-linked immunoassay and immunohistochemistry. In addition, HSV-HGF introduction around the damaged nerve significantly accelerated recovery of function of the facial nerve. These data suggest a possible role of HGF in promoting facial nerve regeneration after nerve damage. Furthermore, this viral delivery method may be applied clinically for many types of severe facial palsy during facial nerve decompression surgery.

Retrograde viral transduction of cortical pyramidal neurons from the spinal cord.

PURPOSE: The primary motor pathway, the corticospinal tract, is a major target for spinal cord regeneration studies. One way of improving the regeneration of corticospinal axons is to introduce regeneration-associated genes into cortical motor neurons using viral vector delivery. METHODS: We used an engineered Herpes Simplex virus (HSV1) with the EF1alpha promoter encoding either LacZ or GFP to transduce cortical neurons through retrograde transport following the injection of vector into adult rat striatum or spinal cord. After three-days to one-month post-injection, sections of brain and spinal cord were viewed with fluorescence microscopy or processed for LacZ histochemistry. RESULTS: Many layer V motor cortical neurons were transduced following striatal injections. These were not corticospinal neurons as they were not fluorogold-labelled following tracer injection into spinal cord. Corticospinal neurons in both hemispheres were, however, transduced following direct vector injections into the dorsal column of spinal cord, yielding 250-400 transduced corticospinal neurons per animal. No non-pyramidal neurons or thalamic neurons were transduced by spinal injections. CONCLUSIONS: Therefore, this HSV1.EF1alpha vector is highly effective for the transduction of corticospinal neurons without direct injection into the brain and could be used to introduce regeneration-relevant genes into these neurons with the aim of regenerating the corticospinal tract.

Cell type specific repression of the varicella zoster virus immediate early gene 62 promoter by the cellular Oct-2 transcription factor.

The cellular transcription factor Oct-2.1 has previously been shown to repress the transactivation of the varicella zoster virus (VZV) immediate early gene promoter by viral transactivators but not to inhibit its basal activity. In the case of the related virus herpes simplex virus (HSV), the effect of Oct-2 on the IE promoters has been shown to be cell type specific and to differ between the different alternatively spliced forms of Oct-2. Here we show that as well as Oct-2.1, the Oct-2.4 and 2.5 isoforms which are expressed in neuronal cells can inhibit transactivation of the VZV immediate early promoter regardless of the cell type used. In contrast, all the isoforms of Oct-2 can inhibit basal activity of the VZV promoter in neuronal cells but not in other cell types indicating that this effect is cell type specific. These effects are discussed in terms of the differential regulation of latent infections with HSV or VZV in dorsal root ganglia.

Viral vectors for gene therapy in Parkinson's disease.

The ability of transplanted neurons from aborted foetuses to produce some therapeutic benefit in Parkinson's disease makes this disease an obvious target for the development of gene therapy procedures which involve delivering the same factors as are provided by the foetal neurons but using a reagent which could be produced in large amounts in a standardised manner. This approach could involve both the delivery of the gene encoding tyrosine hydroxylase to boost dopamine production or the delivery of genes encoding neurotrophic factors such as GDNF to promote the survival of dopaminergic neurons. A variety of different viral and non-viral methods for achieving such gene delivery has been described. These are discussed together with the particular advantages of herpes simplex virus-based vectors which have the potential to deliver multiple therapeutic genes in a single virus vector.

Herpes simplex virus vectors for the nervous system.

Herpes simplex virus type 1 (HSV1) has a number of properties which could potentially be exploited in the development of vectors for the delivery of genes to the nervous system. These include a natural tropism for neurons, a large viral genome allowing the insertion of multiple exogenous genes, and the ability to establish asymptomatic life-long latent infections. Despite these inherent advantages, the development of HSV vectors successfully exploiting all these properties has been problematical. This has mainly been due to either vector toxicity or an inability to maintain transgene expression in the long term. Recent progress towards overcoming these problems and several applications of the technology are discussed.

A gamma34.5 mutant of herpes simplex 1 causes severe inflammation in the brain.

A number of viral vectors are currently being evaluated as potential gene therapy vectors for gene delivery to the brain. As well as evaluating their ability to express a transgene for extended periods of time it is also essential to examine any cytotoxic immune response to such vectors as this may not only limit transgene expression but also cause irreparable harm. This work describes the effect of inoculating a gamma34.5 mutant of herpes simplex type 1 (1716lacZ) into the brain of different strains of rats and mice. Animals were monitored for weight loss and signs of illness, and their brains were evaluated for inflammation, beta-galactosidase expression and recoverable infectious virus. We report that there is (i) a powerful immune response consisting of an early non-specific phase and a later presumably T-cell-mediated phase; (ii) significant weight loss in some animals strains accompanied by severe signs of clinical illness and (iii) transient reporter gene expression in all animal strains examined. To be useful for gene therapy we suggest this virus requires further modification, it should be tested in several animal strains and the dose of virus used may be critical in order to limit damage.

Gene delivery to rat enteric neurons using herpes simplex virus-based vectors.

Neurons of the enteric (gut) nervous system can be cultured in vitro and readily survive transplantation into the brain making close connections with host neurons. As such, they could potentially be used to deliver therapeutic gene products to the brain after transduction with appropriate genes in culture. Here the authors report the first example of gene delivery to such cultured neurons using herpes simplex virus based vectors. They show that viruses lacking the immediate early gene encoding ICP27 (which are unable to replicate lytically) can efficiently deliver a marker gene to enteric neurons without producing extensive cellular damage. In contrast, viruses lacking only the viral neurovirulence factor encoded by ICP34.5 are inefficient in gene delivery, and produce extensive cellular damage, although they cannot replicate lytically in enteric neurons. A virus lacking both ICP27 and ICP34.5, however, produces less cellular damage than one lacking only ICP27, and is as efficient in gene transfer, whereas inactivation of VMW65 reduces toxicity further. The identification of this virus as a safe and efficient gene delivery vector for enteric neurons paves the way for the eventual delivery of therapeutic genes and subsequent transplantation of engineered neurons into the CNS.

Focal cerebral ischaemia increases the levels of several classes of heat shock proteins and their corresponding mRNAs.

The induction of focal cerebral ischaemia in rats by middle cerebral artery occlusion has previously been shown to increase, over time, the mRNA levels of the heat shock proteins (HSPs) 27 and 70. However, the levels of HSP90 mRNA remain constant. In contrast, during global ischaemia, HSP70 and HSP90 mRNA levels are both raised, particularly in the CA1 neurons in the hippocampus, an area that is resistant to the insult in comparison to the surrounding regions. HSP27 mRNA is raised in the neuroglia in the subregions of the hippocampus. However, the protein levels of HSP27, 70 and 90 have not been characterised in focal ischaemia. With this data in mind, we have carried out a comparative study of HSP27, 56, 60, 70 and 90 mRNA and protein levels during focal cerebral ischaemia in rats, up to 24 h post-occlusion. We have shown that HSP70 and HSP27 mRNA levels are increased and also that HSP60 mRNA levels (which had also not previously been characterised in this model of focal ischaemia) are significantly raised. HSP90 and HSP56 mRNAs were not significantly elevated. On Western blot analysis, the inducible HSP72 protein was first detected at 8 h post-occlusion, HSP27 protein was detected only at 24 h post-occlusion and HSP60 protein, although constitutive, appeared to increase at 24 h post-occlusion. HSP56 protein levels appeared to rise on the occluded side, but HSP90 protein levels remained constant.

The degree of protection provided to neuronal cells by a pre-conditioning stress correlates with the amount of heat shock protein 70 it induces and not with the similarity of the subsequent stress.

A mild thermal stress protects primary cultures of dorsal root ganglion (DRG) neurons against a subsequent lethal heat stress as well as to a lesser extent against a subsequent lethal ischaemia. In contrast, a mild ischaemic stress protects DRG neurons only against a subsequent severe thermal stress and not against severe ischaemia. A greater induction of heat shock protein (hsp) synthesis was observed in these cells following mild temperature stress compared to mild ischaemia. This suggests that the protective effect observed is dependent on hsp synthesis resulting in the observed cross-protective effect and does not involve a particular pre-stress specifically protecting against a subsequent, more severe application of the same stress. Moreover, a particular level of hsp induction produces a better protective effect against lethal heat stress than against lethal ischaemia.

Adeno-associated and herpes simplex viruses as vectors for gene transfer to the corneal endothelium.

PURPOSE: We examined the efficacy and cytopathogenicity of adeno-associated (AAV) and herpes simplex viruses (HSV) as vectors for gene transfer to corneal endothelial cells (CECs). METHODS: Recombinant AAV and HSV were examined for their ability to deliver a lacZ histochemical marker gene to whole-thickness rabbit and human corneas ex vivo. Transgene expression was detected with histochemistry and quantified by a colorimetric assay. RESULTS: Rabbit and human corneas transduced with AAV showed increasing numbers of cells expressing marker gene over a 3- to 4-week period. Using 2.5 x 10(6) or 1.5 x 10(7) infective units for rabbit and human corneal specimens, respectively, approximately 2% of CECs expressed the reporter gene. HSV (10(6) plaque-forming units/specimen) transduced approximately 5% of rabbit and human CECs but showed cytotoxicity. In contrast to the duration of recombinant AAV-mediated lacZ expression, recombinant HSV expression was maximal at day 1 and declined to low levels at day 7. CONCLUSION: AAV is a promising vector, but its usefulness for corneal transduction is currently limited by the technical difficulties preparing high titres. The HSV vector examined is efficient but needs further genetic modification to prolong transgene expression and reduce its toxicity.

HSV Mutagenesis.

Herpes genomes are large and complex, with many interactions among herpes encoded proteins, herpes DNA and RNA, and the host cell. These interactions begin as the virus enters the cell, and continue as the decision for latency or lytic replication is made. Correctly regulated gene expression then allows herpes genes to be expressed in a temporally regulated manner and to subvert the host cell metabolism in favor of virus production. Finally, infectious progeny virions are assembled and released. Exploring the function of the many herpes-encoded proteins and the mechanisms to control their expression during these processes thus requires a fine dissection of the herpes genome, so as to allow protein-coding regions to be linked with function and control DNA regions to be identified.

Development of a second-generation oncolytic Herpes simplex virus expressing TNFalpha for cancer therapy.

BACKGROUND: Tumour necrosis factor alpha (TNFalpha) therapy is a promising anti-cancer treatment when combined with radiotherapy due to its potent radio sensitising effects, but systemic toxicity has limited its clinical use. Previously, non-replicative adenovirus vectors have been used to deliver TNFalpha directly to the tumour, including under the control of a radiation sensitive promoter. Here, we have used an ICP34.5 deleted, oncolytic herpes simplex virus (HSV) for delivery to increase expression levels and spread through the tumour, and the use of the US11 true late HSV promoter to limit expression to where the virus replicates, i.e. selectively in tumour tissue. METHODS: TNFalpha expression under the CMV or US11 promoter was compared on cell lines CT26, BHK and Fadu. To further compare the activities of the promoters, expression of human TNFalpha was analysed in the presence and absence of acyclovir--an inhibitor of viral DNA replication and on HSV/ICP34.5- non-permissive cell line 3T6. The in vivo efficacy and toxicity of TNFalpha viruses were compared using A20 double flank tumour model in Balb/C mice and Fadu tumour model in nude mice. RESULTS: The results demonstrated that the US11 promoter significantly reduced and delayed TNFalpha expression as compared to use of the CMV promoter, especially in non-permissive cells or in the presence of acyclovir. Despite the reduced and more selective expression levels, US11 driven TNFalpha showed improved anti-tumour effects compared to CMV driven TNFalpha, and without the toxic side effects. CONCLUSIONS: This approach is therefore beneficial in increasing localised TNFalpha expression as compared to the use of non-replicative approaches, and combines the effects of TNFalpha with oncolytic virus replication which is expected to further enhance the efficacy of radiotherapy in a combined treatment approach.

DsRNA cloning and diagnosis of beet pseudo-yellows virus by PCR and nucleic acid hybridization.

DsRNA has been extracted from beet pseudo-yellows virus infected cucumber plants, purified to homogeneity, and cDNA clones to it produced. The clones are of insufficient sensitivity to detect infection-specific RNA in dot and northern blots of crude nucleic acid extracts. However, a knowledge of the sequence of these clones has been used to synthesize oligonucleotides that have been used for polymerase chain reaction amplification of specific sequences from both purified dsRNA and from infected plants and used as a previously unavailable highly sensitive diagnostic probe. The method of cDNA synthesis has been shown to be generally applicable to some other plant viral dsRNAs and should be of use in the production of cDNA clones when virion RNA is unavailable.

Beet pseudo-yellows virus is an authentic closterovirus.

We have previously used RT-PCR to amplify a 251 base pair fragment diagnostic for beet pseudo-yellows virus (BPYV) infection from several BPYV infected samples from Europe and the USA. This has now been shown by sequencing to represent a fragment of a heat shock protein (HSP)70 gene. Sequence analysis of the fragment revealed that it is closely similar to the C-terminal sequence of the HSP70 homologue gene of lettuce infectious yellows virus, a definitive closterovirus, and also has obvious sequence similarities to the HSP70 genes of the other previously sequenced closteroviruses citrus tristeza virus and beet yellows virus. This observation confirms that BPYV is an authentic closterovirus with a wide geographical distribution.

Viral vectors in the treatment of Parkinson's disease.

Parkinson's disease is an obvious target for the development of gene therapy procedures which could involve both the delivery of the gene encoding tyrosine hydroxylase to boost dopamine production or the delivery of genes encoding neurotrophic factors such as GDNF to promote the survival of dopaminergic neurons. A variety of different viral and nonviral methods for achieving such gene delivery are described together with the particular advantages of herpes simplex virus-based vectors which have the potential to deliver multiple therapeutic genes in a single virus vector.

The tobacco necrosis virus p7a protein is a nucleic acid-binding protein.

The two centrally located open reading frames (ORFs) of necroviruses may, by analogy with the similarly located and related ORFs of carmoviruses, be expected to have a function in virus movement in plants. In the case of tobacco necrosis virus (TNV) strain D these proteins both have a molecular mass of approximately 7 kDa and are thus known as p7a and p7b. We overexpressed p7a in Escherichia coli, separated it from bacterial proteins and renatured it on gels, and showed that p7a was able to bind single-stranded RNA and single-stranded DNA, but was unable to bind double-stranded DNA. These protein-nucleic acid complexes were stable at moderately high salt concentrations. Protein p7b could not be expressed in a number of bacterial systems. We speculate that in TNV, unlike some other viruses which encode a single movement protein with separate functional domains for RNA binding and plasmodesmatal targeting, p7a and p7b may respectively provide these functions on separate proteins.

Altered dinucleotide content within the latently transcribed regions of the DNA of alpha herpes viruses--implications for latent RNA expression and DNA structure.

The alphaherpesviruses establish latent infections in sensory neuronal cells, during which a number of latency-associated RNA transcripts (LATs) are produced. The reasons for the production of the LATs, however, and the mechanism by which the LAT region of the genome remains active during latency are unknown. Here we have analysed alphaherpesvirus sequences in an attempt to assess any differences between latently active regions of the genomes and the genomes as a whole which might be necessary for this differential expression pattern or for the LAT function, whatever that might be. We show that the LAT regions of all of the four alphaerpesviruses examined exhibit a previously unidentified and marked increase in CC + GG dinucleotides within the region and that three of the four viruses examined also show a marked decrease in CG + GC dinucleotides. The CC + GG increase was shown to occur throughout the LAT regions of these viruses, but the GC + CG decrease was shown to be confined only to LATs which are thought to be expressed as introns, and also to the intron sequences of the IE1 genes (at least for herpes simplex viruses 1 and 2), which overlap the LAT region. Bovine herpesvirus, the virus which did not show the GC + CG reduction in its LAT region but did show the GG + CC increase, is thought not to express LATs by a mechanism involving splicing, again indicating that the CG + GC reduction may be an intron-related phenomenon and also further suggesting that the two effects of CG + GC decrease and GG + CC increase may be independent of one another. Possible reasons for these unusual dinucleotide frequencies within the latently active regions of the alphaherpesviruses relating to DNA and RNA structure and to LAT function are discussed.

The Nogo receptor, its ligands and axonal regeneration in the spinal cord; a review.

At least three proteins present in CNS myelin, Nogo, MAG and OMgp are capable of causing growth cone collapse and inhibiting neurite outgrowth in vitro. Surprisingly, Nogo and OMgp are also strongly expressed by many neurons (including neocortical projection cells). Nogo expression is increased by some cells at the borders of CNS lesion sites and by cells in injured peripheral nerves, but Nogo and CNS myelin are largely absent from spinal cord injury sites, which are none the less strongly inhibitory to axonal regeneration. Nogo is found on growing axons during development, suggesting possible functions for neuronal Nogo in axon guidance. Although Nogo, MAG and OMgp lack sequence homologies, they all bind to the Nogo receptor (NgR), a GPI-linked cell surface molecule which, in turn, binds p75 to activate RhoA. NgR is strongly expressed by cerebral cortical neurons but many other neurons express NgR weakly or not at all. Some neurons, such as DRG cells, respond to Nogo and CNS myelin in vitro although they express little or no NgR in vivo which, with other data, indicates that other receptors are available for NgR ligands. NgR expression is unaffected by injury to the nervous system, and there is no clear correlation between NgR expression by neurons and lack of regenerative ability. In the injured spinal cord, interactions between NgR and its ligands are most likely to be important for limiting regeneration of corticospinal and some other descending tracts; other receptors may be more important for ascending tracts. Antibodies to Nogo, mainly the poorly-characterised IN-1 or its derivatives, have been shown to enhance recovery from partial transections of the spinal cord. They induce considerable plasticity from the axons of corticospinal neurons, including sprouting across the midline and, to a limited extent, regeneration around the lesion. Regeneration of corticospinal axons induced by Nogo antibodies has not yet been demonstrated after complete transections or contusion injuries of the spinal cord. It is not clear whether antibodies against Nogo act on oligodendrocytes/myelin or by binding to neuronal Nogo, or whether they can stimulate regeneration of ascending axons in the spinal cord, most of which express little or no NgR. Despite these uncertainties, however, NgR and its ligands offer important new targets for enhancing plasticity and regeneration in the nervous system.