The concept is if it is possible to repair damage by providing appropriate cues to the endogenous neural staminal cells (NSCs) and progenitors; cues that are not available or insufficient in the injured tissue. The therapeutic applicability of this system was proved in a model of neuronal loss, the hippocampal sclerosis induced by prolonged generalized seizures. In this model, an epileptogenic insult (the episode of prolonged seizures) causes a damage pattern in the hippocampus that closely mimics the one observed in many patients affected by the most common adult epileptic syndrome [43Kann O, Kovacs R, Njunting M, et al. Metabolic dysfunction during neuronal activation in the ex vivo hippocampus from chronic epileptic rats and humans Brain 2005; 128: 2396-407.]. In time, animals begin to display spontaneously recurrent seizures, i.e. they become truly epileptic, again reproducing the situation observed in patients [44Lehmann TN, Gabriel S, Kovacs R, et al. Alterations of neuronal connectivity in area CA1 of hippocampal slices from temporal lobe epilepsy patients and from pilocarpine-treated epileptic rats Epilepsia 2000; 41(Suppl 6): S190-4., 45Tetz LM, Rezk PE, Ratcliffe RH, et al. Development of a rat pilocarpine model of seizure/status epilepticus that mimics chemical warfare nerve agent exposure Toxicol Ind Health 2006; 22: 255-66.]. Paradiso and co-workers demonstrated that recombinant HSV-1-based vectors expressing a combination of two NTFs, fibroblast growth factor-2 (FGF-2) and brain-derived neurotrophic factor (BDNF), increased survival and proliferation of freshly isolated neural progenitors and favoured their differentiation into neurons in vitro. These vectors were tested in vivo, in the pilocarpine model of status epilepticus-induced neurodegeneration and epileptogenesis. When injected in the hippocampus 3 days after status epilepticus, FGF-2/BDNF expressing vectors partially repaired neuronal damage and prevented the occurrence of spontaneous seizures. Thus, viral vector-mediated supplementation of FGF-2 and BDNF promotes neurogenesis and repair of an existing neuronal damage and these effects are disease-modifying in epilepsies associated with hippocampal sclerosis, demonstrating the feasibility of use of HSV vectors expressing NTFs to provide recovery from damage and to prevent the development of epilepsy [35Paradiso B, Marconi P, Zucchini S, et al. Localized delivery of fibroblast growth factor-2 and brain-derived neurotrophic factor reduces spontaneous seizures in an epilepsy model Proc Natl Acad Sci USA 2009; 106: 7191-6.].

One of the most important human demyelinating diseases of unknown aetiology is multiple sclerosis (MS), an autoimmune-mediated inflammatory disease of the CNS with inflammatory infiltrates containing auto-reactive T cells and a multitude of pathogenic nonspecific lymphocytes that might benefit from anti-inflammatory therapies [46Fernandez O. Combination therapy in multiple sclerosis J Neurol Sci 2007; 259: 95-103., 47Compston A. The basis for treatment in multiple sclerosis Acta Neurol Scand Suppl 2006; 183: 41-7.]. Furlan and colleagues have used viral vectors expressing immune-modulators to treat experimental autoimmune encephalomyelitis (EAE), which is a mouse model for MS, showing the therapeutic efficacy of non-replicating HSV-1-derived vectors expressing anti-inflammatory genes, such as cytokine interleukin-4 (IL-4) [48Furlan R, Brambilla E, Ruffini F, et al. Intrathecal delivery of IFN-gamma protects C57BL/6 mice from chronic-progressive experimental autoimmune encephalomyelitis by increasing apoptosis of central nervous system-infiltrating lymphocytes J Immunol 2001; 167: 1821-9.] or cytokine interleukin-1 receptor antagonist (IL-1ra) [34Furlan R, Bergami A, Brambilla E, et al. HSV-1-mediated IL-1 receptor antagonist gene therapy ameliorates MOG(35-55)-induced experimental autoimmune encephalomyelitis in C57BL/6 mice Gene Ther 2007; 14: 93-8., 49Martino G, Poliani PL, Marconi PC, Comi G, Furlan R. Cytokine gene therapy of autoimmune demyelination revisited using herpes simplex virus type-1-derived vectors Gene Ther 2000; 7: 1087-93.]. They have demonstrated that, after disease onset, CNS administration of HSV-1 defective recombinant vectors expressing IL-4 or IL-1ra genes into Biozzi AB/H mice stopped the progression of relapsing–remitting form of EAE. The treated mice showed a shorter duration of the first EAE attack, a longer inter-relapse period, and a reduction in the severity and duration of the relapse. The results obtained by this group have revealed an in situ modulation of the cytokine/chemokine circuits, demonstrating that the local administration of anti-inflammatory cytokines by viral vectors can be effective in the preventive treatment of chronic EAE [34Furlan R, Bergami A, Brambilla E, et al. HSV-1-mediated IL-1 receptor antagonist gene therapy ameliorates MOG(35-55)-induced experimental autoimmune encephalomyelitis in C57BL/6 mice Gene Ther 2007; 14: 93-8.].

Accumulation of insoluble aggregates of amyloid-β peptide (Aβ), a cleavage product of amyloid precursor protein (APP), is thought to be central to the pathogenesis of Alzheimer’s disease (AD). Consequently, down-regulation of APP, or enhanced clearance of Aβ, represent possible therapeutic strategies for AD. Hong CS and colleagues [36Hong CS, Goins WF, Goss JR, Burton EA, Glorioso JC. Herpes simplex virus RNAi and neprilysin gene transfer vectors reduce accumulation of Alzheimer's disease-related amyloid-beta peptide in vivo Gene Ther 2006; 13: 1068-79.] have generated replication-defective HSV vectors that inhibit Aβ accumulation, both in vitro and in vivo. In cell culture, HSV vectors expressing either (i) short hairpin RNA (siRNA) directed to the APP transcript (HSV-APP/shRNA), or (ii) neprilysin, an endopeptidase that degrades Aβ (HSV-neprilysin), substantially inhibited accumulation of Aβ. To determine whether these vectors showed similar activity in vivo, it was developed a novel mouse model, in which overexpression of a mutant form of APP in the hippocampus, using a lentiviral vector (LV-APPSw), resulted in rapid Aβ accumulation. Co-inoculation of LV-APPSw with each of the HSV vectors showed that either HSV-APP/shRNA or HSV-neprilysin inhibited Aβ accumulation in this model, whereas an HSV control vector did not. These studies demonstrate the utility of HSV vectors for reducing Aβ accumulation in the brain, thus providing useful tools to clarify the role of Aβ in AD that may facilitate the development of novel therapies for this important disease.

Some brain diseases that are amenable to gene therapy are localized to particular region of the brain; example of these type of disorders include Parkinson’s disease. Using a replication-defective HSV vector expressing either glial cell derived neurotrophic factor (GDNF) or the antiapoptotic peptide bcl-2, it has been shown that direct inoculation of the HSV vector into the substantia nigra can be used to protect rodents from 6-hydroxidopamine-induced degeneration of dopaminergic neurons [50Yamada M, Oligino T, Mata M, et al. Herpes simplex virus vector-mediated expression of Bcl-2 prevents 6-hydroxydopamine-induced degeneration of neurons in the substantia nigra in vivo Proc Natl Acad Sci USA 1999; 96: 4078-83., 51Natsume A, Mata M, Goss J, et al. Bcl-2 and GDNF delivered by HSV-mediated gene transfer act additively to protect dopaminergic neurons from 6-OHDA-induced degeneration Exp Neurol 2001; 169: 231-8.]. Using latency associated promoter (LAP2) to drive GDNF expression in the replication-defective vector prolonged biologically active transgene expression, over the course of many months, has been obtained [52Puskovic V, Wolfe D, Goss J, et al. Prolonged biologically active transgene expression driven by HSV LAP2 in brain in vivo Mol Ther 2004; 10: 67-75.]. In another study, it has been investigated the neuroprotective effect of erythropoietin (EPO) in a rodent model of Parkinson disease [37Puskovic V, Wolfe D, Wechuck J, et al. HSV-mediated delivery of erythropoietin restores dopaminergic function in MPTP-treated mice Mol Ther 2006; 14: 710-5.]. The effects of vector-produced EPO were similar in magnitude to the effects of vector-mediated GDNF in the same model. These results demonstrate that vector-mediated EPO production may be used to reverse dopaminergic neurodegeneration in the face of continued toxic insult.

Neuropathy is a common, untreatable complication of both type 1 and type 2 diabetes. In animal models peptide neurotrophic factors can be used to protect against the development of neuropathy, but the combination of short half-life and off-target effects of these potent pleiotropic peptides has limited translation to human therapy. Nerve growth factor (NGF) is probably the most extensively studied trophic factor in diabetic neuropathy. NGF levels are reduced in diabetic nerve, and though NGF receptor expression is normal in streptozotocin (STZ)-induced diabetic nerve there is a marked decrease in receptor saturation and concomitant reduced retrograde axonal transport of NGF. Other trophic factors that have been shown to have a protective effect in diabetic neuropathy models in rodent include: brain derived neurotrophic factor (BDNF), neurotrophin 3 (NT-3); ciliary neurotrophic factor (CNF), vascular endothelial growth factor (VEGF), IL-6, glial cell derived neurotrophic factor (GDNF), fibroblast growth factor (FGF) and erythropoietin. A detailed review on the therapeutic effects of the above receptor factors has been published [53Calcutt NA, Jolivalt CG, Fernyhough P. Growth factors as therapeutics for diabetic neuropathy Curr Drug Targets 2008; 9: 47-59.]. Results suggesting a protective effect of NGF in patients with diabetic neuropathy were obtained in a phase 2 human trial but the following phase 3 prospective randomized control trial of efficacy NGF was found to be ineffective in preventing the progression of neuropathy [54Apfel SC, Schwartz S, Adornato BT, et al. Efficacy and safety of recombinant human nerve growth factor in patients with diabetic polyneuropathy: A randomized controlled trial. rhNGF Clinical Investigator Group JAMA 2000; 284: 2215-1.]. Number of potential pitfalls can be identified that may have contributed to the failure of NGF treatment in this trial. The most obvious is the dose-related effect and that the treatment needs to be effective for a prolonged period of time.

Gene transfer is a promising strategy that might circumvent these limitations [55Mata M, Hao S, Fink DJ. Applications of gene therapy to the treatment of chronic pain Curr Gene Ther 2008; 8: 42-8.]. Replication-defective HSV vectors have been tested in several different models of neuropathy leading up to studies in diabetes [56Chattopadhyay M, Goss J, Lacomis D, et al. Protective effect of HSV-mediated gene transfer of nerve growth factor in pyridoxine neuropathy demonstrates functional activity of trkA receptors in large sensory neurons of adult animals Eur J Neurosci 2003; 17: 732-40.-61Mata M, Chattopadhyay M, Fink DJ. Gene therapy for the treatment of diabetic neuropathy Curr Diab Rep 2008; 8: 431-6.]. In an experimental model of type 1 diabetes in STZ mice, subcutaneous inoculation of replication-defective HSV expressing VEGF was able to prevent the reduction in foot sensory nerve amplitude characteristic of diabetes [58Chattopadhyay M, Krisky D, Wolfe D, et al. HSV-mediated gene transfer of vascular endothelial growth factor to dorsal root ganglia prevents diabetic neuropathy Gene Ther 2005; 12: 1377-84.]. Moreover, in a model of diabetic cystopathy, HSV vector-mediated NGF gene therapy has been proved useful to restore decreased NGF expression in the bladder and bladder afferent pathways, thereby improving hypoactive bladder function in diabetes [62Sasaki K, Chancellor MB, Goins WF, et al. Gene therapy using replication-defective herpes simplex virus vectors expressing nerve growth factor in a rat model of diabetic cystopathy Diabetes 2004; 53: 2723-30.]. In a further study aimed to determine whether an HSV latency promoter can prolong expression of neurotrophin 3 (NT-3), it has been shown that the continuous production of NT-3 by Lap2-driven long-term expression of the transgene from an HSV vector protects against progression of diabetic neuropathy in mice [31Chattopadhyay M, Mata M, Goss J, et al. Prolonged preservation of nerve function in diabetic neuropathy in mice by herpes simplex virus-mediated gene transfer Diabetologia 2007; 50: 1550-8.].

Over the past several years, studies of the mechanisms that are involved in the development of chronic pain have created novel information that can lead to identify multiple points of intervention to treat this pathological condition. On the basis of this knowledge, HSV-based gene therapy approaches have been developed to locally express products that block pain transmission or reverse the chronic pain state [40Glorioso JC, Fink DJ. Herpes vector-mediated gene transfer in the treatment of chronic pain Mol Ther 2009; 17: 13-8., 63Goins WF, Goss JR, Chancellor MB, et al. Herpes simplex virus vector-mediated gene delivery for the treatment of lower urinary tract pain Gene Ther 2009; 16: 558-69.-66Westlund KN. Gene therapy for pancreatitis pain Gene Ther 2009; 16: 483-92.]. Preclinical studies on pain animal models have demonstrated in vivo the capacity of these vectors to effectively transfer genes into the dorsal root ganglia (DRG) neurons following direct pancreatic inoculation or subcutaneous inoculation to efficiently express and release inhibitory neurotransmitters or anti-inflammatory peptides that can be used to modulate pain-related behaviours and provide a therapeutic effect in models of poly-neuropathy and chronic regional pain [40Glorioso JC, Fink DJ. Herpes vector-mediated gene transfer in the treatment of chronic pain Mol Ther 2009; 17: 13-8., 67Liu J, Wolfe D, Hao S, et al. Peripherally delivered glutamic acid decarboxylase gene therapy for spinal cord injury pain Mol Ther 2004; 10: 57-66., 68Lee JY, Fink DJ, Mata M. Vector-mediated gene transfer to express inhibitory neurotransmitters in dorsal root ganglion reduces pain in a rodent model of lumbar radiculopathy Spine 2006; 31: 1555-8.]. Opioid receptors are found presynaptically on the terminals of primary nociceptive afferents in the spinal cord, and post synaptically on second-order neurons in the dorsal horn. Activation of receptors, naturally by endogenous ligands enkephalin or endomorphin, or therapeutically by opiate drugs such as morphine, inhibits pain-related neurotransmission at the spinal level. The first vectors to be used in models of pain were deleted for accessory viral functions (e.g. thymidine kinase, tk). Recombinant HSV defective in tk can be propagated in culture and will replicate in skin, but are unable to replicate in the DRG and are thus forced into a pseudo-latent state. The efficacy of HSV-mediated gene transfer of enkephalin has been tested in several different models of pain in rodents [69Wilson SP, Yeomans DC, Bender MA, et al. Antihyperalgesic effects of infection with a preproenkephalin-encoding herpes virus Proc Natl Acad Sci USA 1999; 96: 3211-6., 70Antunes Bras JM, Epstein AL, Bourgoin S, et al. Herpes simplex virus 1-mediated transfer of preproenkephalin A in rat dorsal root ganglia J Neurochem 1998; 70: 1299-303.]. Expression of enkephalin from TK-negative vectors did not only reduce pain-related behaviours, but also prevented cartilagine and bone distruction in the inflamed joint. Similar effect were subsequentely demonstrated using a nonreplicating HSV vector deleted for both copies of the essential HSV IE gene ICP4 [71Goss JR, Mata M, Goins WF, et al. Antinociceptive effect of a genomic herpes simplex virus-based vector expressing human proenkephalin in rat dorsal root ganglion Gene Ther 2001; 8: 551-6.]. The observations regarding reinoculation of the vector, which have been repeated in several different model of pain and with different transgene products, indicate the absence of any significant immune response to vector inoculation in rodent. Studies of the enkephalin-expressing HSV vectors have been extended to primates [72Yeomans DC, Lu Y, Laurito CE, et al. Recombinant herpes vector-mediated analgesia in a primate model of hyperalgesia Mol Ther 2006; 13: 589-97.] and provide proof-of-principle evidence that HSV vector-mediated delivery of enkephalin can provide an analgesic effect and set the stage for a human trial to treat chronic pain using HSV vector-expressing enkephalin. The clinical-grade replication-defective ICP4 and ICP27 deleted vector engineered to express preproenkephalin was termed NP2. This vector entered in phase 1 clinical trial, enrolling patients with cancer-associated pain, and based on the predefined parameters of the toxicology study was well tolerated with no significant toxicity [39Wolfe D, Mata M, Fink DJ. A human trial of HSV-mediated gene transfer for the treatment of chronic pain Gene Ther 2009; 16: 455-60.].

Animal pancreatitis models have been used to test innovative preclinical, site-specific gene therapeutic interventions [65Yang H, McNearney TA, Chu R, et al. Enkephalin-encoding herpes simplex virus-1 decreases inflammation and hotplate sensitivity in a chronic pancreatitis model Mol Pain 2008; 4: 8., 66Westlund KN. Gene therapy for pancreatitis pain Gene Ther 2009; 16: 483-92.]. The efficacy of pre- and post-treatment with a replication-defective HSV-1 vector construct encoding human preproenkephalin gene (HSV-Enk) to bring overexpressed opioids directly to pancreatic tissue was tested. Reduced pain-related behaviours, inflammatory and cellular activation responses in rats with acute and chronic pancreatitis were demonstrated after direct injection of the viral vector into the pancreas by laparatomy. As an important safety issue, in this experimental model there was no evidence of spread of HSV-1 centrally or peripherally other than into the appropriate level DRG. HSV-1 vectors that express glutamic acid decarboxylase (GAD) have been shown to be more effective than the opioid peptide in neuropathic pain [67Liu J, Wolfe D, Hao S, et al. Peripherally delivered glutamic acid decarboxylase gene therapy for spinal cord injury pain Mol Ther 2004; 10: 57-66., 68Lee JY, Fink DJ, Mata M. Vector-mediated gene transfer to express inhibitory neurotransmitters in dorsal root ganglion reduces pain in a rodent model of lumbar radiculopathy Spine 2006; 31: 1555-8.]. Defective HSV-1-derived vectors expressing anti-inflammatory cytokines, such as IL-4 or IL-10 [39Wolfe D, Mata M, Fink DJ. A human trial of HSV-mediated gene transfer for the treatment of chronic pain Gene Ther 2009; 16: 455-60., 73Zhou Z, Peng X, Hao S, Fink DJ, Mata M. HSV-mediated transfer of interleukin-10 reduces inflammatory pain through modulation of membrane tumor necrosis factor alpha in spinal cord microglia Gene Ther 2008; 15: 183-90.], have been deeply studied to examine the involvement of cytokines in the development of inflammatory pain. Mata and colleagues have demonstrated, in a rat model of inflammatory pain, that expression of IL-10 by a HSV-1 vector in DRG prevents activation of p38 mitogen-activated protein kynase (p38 MAPK) and expression of full-length membrane-spanning tumour necrosis factor-α (mTNFα) in dorsal horn and spinal cord, suggesting the involvement of TNFα in the development of inflammatory and neuropathic pain [55Mata M, Hao S, Fink DJ. Applications of gene therapy to the treatment of chronic pain Curr Gene Ther 2008; 8: 42-8., 74Hao S, Mata M, Glorioso JC, Fink DJ. Gene transfer to interfere with TNFalpha signaling in neuropathic pain Gene Ther 2007; 14: 1010-6.]. Plans for an efficacy trial with an opioid producing vector in inflammatory pain and with a GAD producing vector in diabetic neurophatic pain are outlined.

Recently, it has been shown silencing in DRG neurons in vivo by vector-mediated delivery of small interfering RNA (shRNA). This study support the utility of HSV vectors for gene silencing in peripheral neurons and the potential application of this technology to the study of nociceptive processes and in pain gene target validation studies [64Anesti AM, Peeters PJ, Royaux I, Coffin RS. Efficient delivery of RNA Interference to peripheral neurons in vivo using herpes simplex virus Nucleic Acids Res 2008; 36: e86.].

The lysosomal storage diseases (LSDs) are caused by genetic defects in lysosomal enzymes that result in the accumulation of substrates in the lysosomes. Over 50 LSDs exist and the group of disease has a collective occurrence of about one in 7,000 [75Meikle PJ, Hopwood JJ, Clague AE, Carey WF. Prevalence of lysosomal storage disorders JAMA 1999; 281: 249-54.]. Therapy of lysosomal storage disorders with neurological involvement, such as Tay-Sachs (TS) disease, requires active hexosaminidase (Hex) A production in the CNS and an efficient therapeutic approach that can act faster than human disease progression. Several therapeutic approaches have been developed that allow to restore the enzymatic activity in many key tissues (kidney, liver, spleen, etc.). However, the reduction of the GM2 ganglioside deposits in the CNS is difficult to achieve since CNS represents a privileged environment, separated from the blood system by the blood-brain barrier (BBB) that is an obstacle to therapy. Martino S. and colleagues [42Martino S, Marconi P, Tancini B, et al. A direct gene transfer strategy via brain internal capsule reverses the biochemical defect in Tay-Sachs disease Hum Mol Genet 2005; 14: 2113-3.] combined the efficacy of a non-replicating HSV-1 vector encoding for the Hex A alpha-subunit (HSV-T0alphaHex) and the anatomic structure of the brain internal capsule to distribute the missing enzyme optimally. With this gene transfer strategy, for the first time, it was possible to re-establish the Hex A activity and totally remove the GM2 ganglioside storage in both injected and controlateral hemispheres, in the cerebellum and spinal cord of TS animal model in the span of one month’s treatment. In this study, no adverse effects were observed due to the viral vector, injection site or gene expression and it has been hypothesized that the same approach could be applied to similar diseases involving an enzyme defect [42Martino S, Marconi P, Tancini B, et al. A direct gene transfer strategy via brain internal capsule reverses the biochemical defect in Tay-Sachs disease Hum Mol Genet 2005; 14: 2113-3.].

Berges B.K. and colleagues [41Berges BK, Yellayi S, Karolewski BA, et al. Widespread correction of lysosomal storage in the mucopolysaccharidosis type VII mouse brain with a herpes simplex virus type 1 vector expressing beta-glucuronidase Mol Ther 2006; 13: 859-69.] have used HSV1716 attenuated vector, an ICP34.5 null mutant, to treat Mucopolysaccharidosis (MPS) VII. MPS VII is caused by a gene deficiency in the lysosomal enzyme β-glucoronidase (GUSB) with consequent accumulation of glycosaminoglycans (GAGs) affecting a number of organ systems. The investigators have demonstrated the capacity of HSV1716 virus expressing GUSB driven by the latency-associated transcript promoter (HSV-1716-LAT-GUSB virus) to correct the lysosomal storage in the adult MPS VII mouse model following intracranial injection. The authors have shown that this neuroattenuated vector was able to establish latency and to express GUSB at a distance from the site of injection and the correction of the lysosomal storage was demonstrated in several brain regions. This result was the consequence of the ability of neuroattenuated vectors to traverse at least one neuronal synapse and achieve gene expression in a secondary neuron followed by the secretory properties of GUSB that can cross-correct a larger brain area.

Two neuroattenuated, ICP4-negative HSV vectors expressing IL-4 or IL-10 have been used as gene therapy approach in the EAE animal model for human MS [76Broberg E, Setala N, Roytta M, et al. Expression of interleukin-4 but not of interleukin-10 from a replicative herpes simplex virus type 1 viral vector precludes experimental allergic encephalomyelitis Gene Ther 2001; 8: 769-7.]. The results of this study demonstrate that local expression of IL-4 from a replication-competent HSV vector precludes sign of EAE, whereas local expression of IL-10 did not have the disease-abolishing effect as did the expression of IL-4, although IL-10 has been shown to be connected to the recovery phase of disease development of EAE. These results confirm the different roles of different Th2-type cytokines expressed during the recovery phase of EAE.

The development of therapeutic strategies to attenuate the neurological symptoms of acute injury is the subject of major research interest. However, effective treatments remain elusive, due at least in part, to the complex cell-to-cell interactions that regulate neuron cell life/death decisions. Therefore, multiple-target strategies to rescue neurons, including those that surround the treated ones, are particularly desirable. At this regard, it has been recently reported that neurons surviving apoptosis through expression of HSV-2 ICP10PK gene (containing a deletion in the protein kinase -PK- domain of the ICP10 gene), delivered by the replication attenuated vector ∆RR, release increased levels of VEGF and FKN that protect uninfected neurons from apoptosis both through neuron-neuron and bidirectional neuron-microglia communications [77Laing JM, Smith CC, Aurelian L. Multi-targeted neuroprotection by the HSV-2 gene ICP10PK includes robust bystander activity through PI3-K/Akt and/or MEK/ERK-dependent neuronal release of vascular endothelial growth factor and fractalkine J Neurochem 2010; 112: 662-76.]. The latter involves increased release of IL-10 and decreased release of TNF-α by the FKN-treated microglia. Further to these considerations, it has been suggested that ∆RR delivered ICP10PK is a multiple-targeted strategy to rescue neurons. It has the distinct advantage that in addition to protecting the infected neurons, it modulates them to release neuroprotective soluble factors in a balanced proportion such as to create a self-propagating cycle of neuronal inputs and release of chemical mediators that inhibit the progression of acute and chronic neurodegeneration through protection of uninfected neurons.

The first generation of these vectors contains mutation in a single gene that restricted their replication to dividing cells. Three such HSV-1 mutants have been constructed: (1) dlsptk containing a deletion in the tk gene [148Martuza RL, Malick A, Markert JM, Ruffner KL, Coen DM. Experimental therapy of human glioma by means of a genetically engineered virus mutant Science 1991; 252: 854-6.]; (2) hrR3 containing an insertion of the E. coli lac-Z gene in the early gene UL39, encoding the large subunit of the viral RR (ICP6) [149Kramm CM, Rainov NG, Sena-Esteves M, et al. Long-term survival in a rodent model of disseminated brain tumors by combined intrathecal delivery of herpes vectors and ganciclovir treatment Hum Gene Ther 1996; 7: 1989-94., 150Jeyaretna DS, Rabkin SD, Martuza RL. Oncolytic herpes simplex virus therapy for peripheral nerve tumors Neurosurg Focus 2007; 22: E4.]; (3) R3616 containing 1 kb deletions in both copies of the γ-34.5 gene, encoding the neurovirulence factor ICP34.5 [151Andreansky S, Soroceanu L, Flotte ER, et al. Evaluation of genetically engineered herpes simplex viruses as oncolytic agents for human malignant brain tumors Cancer Res 1997; 57: 1502-9.-153Dambach MJ, Trecki J, Martin N, Markovitz NS. Oncolytic viruses derived from the gamma34.5-deleted herpes simplex virus recombinant R3616 encode a truncated UL3 protein Mol Ther 2006; 13: 891-.].

TK mutants are highly neuroattenuated, and when used in different mouse models of various nervous system-derived tumour types, showed a slowed tumour growth and prolonged survival. However, clinical trials were not pursued because of (i) undesirable level of toxicity at high titers, and (ii) its TK-negative status made it resistant to traditional anti-herpetic treatments, a major disadvantage should any viral toxicity to arise in treated patients [148Martuza RL, Malick A, Markert JM, Ruffner KL, Coen DM. Experimental therapy of human glioma by means of a genetically engineered virus mutant Science 1991; 252: 854-6., 154Boviatsis EJ, Scharf JM, Chase M, et al. Antitumor activity and reporter gene transfer into rat brain neoplasms inoculated with herpes simplex virus vectors defective in thymidine kinase or ribonucleotide reductase Gene Ther 1994; 1: 323-1.-156Carroll NM, Chase M, Chiocca EA, Tanabe KK. The effect of ganciclovir on herpes simplex virus-mediated oncolysis J Surg Res 1997; 69: 413-7.].

ICP6 mutants have been tested as replicative anticancer agents, alone or in combination with acyclovir/gancyclovir, as the mutants retain their sensitivity to such antivirals. Moreover, the RR^- mutants have been shown to display an increased sensitivity to gancyclovir, compared to the wt virus. These recombinant viruses showed enhanced killing of tumour cells in vitro, and showed improved survival of animals. However, like TK mutants, they can cause fatal encephalitis when used at sufficient dose and were not thought to provide a sufficient margin of safety for testing in humans [149Kramm CM, Rainov NG, Sena-Esteves M, et al. Long-term survival in a rodent model of disseminated brain tumors by combined intrathecal delivery of herpes vectors and ganciclovir treatment Hum Gene Ther 1996; 7: 1989-94., 157Chambers R, Gillespie GY, Soroceanu L, et al. Comparison of genetically engineered herpes simplex viruses for the treatment of brain tumors in a scid mouse model of human malignant glioma Proc Natl Acad Sci USA 1995; 92: 1411-5.].

It has been shown that deletion of ICP34.5, the neurovirulence factor essential for HSV pathogenicity, provides the greatest degree of attenuation of any individual mutation where the virus can still replicate in actively dividing cells. R3616, the prototype HSV-1 deleted in both copies of γ-34.5, had demonstrated attenuated neurovirulence but with maintained anti-glioma activity, and was found to produce no encephalitis in a nude mouse model [157Chambers R, Gillespie GY, Soroceanu L, et al. Comparison of genetically engineered herpes simplex viruses for the treatment of brain tumors in a scid mouse model of human malignant glioma Proc Natl Acad Sci USA 1995; 92: 1411-5.-159Markert JM, Malick A, Coen DM, Martuza RL. Reduction and elimination of encephalitis in an experimental glioma therapy model with attenuated herpes simplex mutants that retain susceptibility to acyclovir Neurosurgery 1993; 32: 597-603.]. The use of γ-34.5 mutated viruses demonstrated considerable antitumor efficacy, combined with a good safety profile, and different versions of HSV ICP34.5-deleted are currently in human clinical trials [160Liu BL, Robinson M, Han ZQ, et al. ICP34.5 deleted herpes simplex virus with enhanced oncolytic, immune stimulating, and anti-tumour properties Gene Ther 2003; 10: 292-303.].

Following preclinical testing with the above-mentioned oncolytic vectors, second generation vectors with multigenic mutations were created. G207 contains deletion in both γ-34.5 loci and a lac-Z gene insertion in the ICP6 gene [161Mineta T, Rabkin SD, Yazaki T, Hunter WD, Martuza RL. Attenuated multi-mutated herpes simplex virus-1 for the treatment of malignant gliomas Nat Med 1995; 1: 938-43.]. These multiple mutations made the reversion to wt highly unlikely and conferred several important safety advantages. Moreover, G207 retains its susceptibility to standard anti-HSV therapies such as acyclovir, since the tk gene is intact. After oncolytic activity and safety evaluation studies in the mouse model [161Mineta T, Rabkin SD, Yazaki T, Hunter WD, Martuza RL. Attenuated multi-mutated herpes simplex virus-1 for the treatment of malignant gliomas Nat Med 1995; 1: 938-43.], G207 neurotoxicity was further evaluated in non-human primates [162Hunter WD, Martuza RL, Feigenbaum F, et al. Attenuated, replication-competent herpes simplex virus type 1 mutant G207: safety evaluation of intracerebral injection in nonhuman primates J Virol 1999; 73: 6319-26.]. These studies allowed to move into phase I clinical trials [163Markert JM, Medlock MD, Rabkin SD, et al. Conditionally replicating herpes simplex virus mutant, G207 for the treatment of malignant glioma: results of a phase I trial Gene Ther 2000; 7: 867-74.], and, presently, enrolment has begun for sequential phases Ib/II trials employing G207 as an anti-tumour agent for malignant gliomas [164Markert JM, Liechty PG, Wang W, et al. Phase Ib trial of mutant herpes simplex virus G207 inoculated pre-and post-tumor resection for recurrent GBM Mol Ther 2009; 17: 199-207., 165Aghi MK, Chiocca EA. Phase ib trial of oncolytic herpes virus G207 shows safety of multiple injections and documents viral replication Mol Ther 2009; 17: 8-9.]. MGH-1 is an oncolytic HSV-1 which shares the same characteristics of G207 and its oncolytic activity was evaluated in preclinical models [155Kramm CM, Chase M, Herrlinger U, et al. Therapeutic efficiency and safety of a second-generation replication-conditional HSV1 vector for brain tumor gene therapy Hum Gene Ther 1997; 8: 2057-68., 166Jacobs A, Tjuvajev JG, Dubrovin M, et al. Positron Emission tomography-based imaging of transgene expression mediated by replication-conditional, oncolytic herpes simplex virus type 1 mutant vectors in vivo Cancer Res 2001; 61: 2983-95.].

Almost simultaneously, HSV1716, derived from the parent wt strain HSV-1 17+ in which both the copies of γ-34.5 have been deleted, also underwent clinical trials to evaluate its toxicity in patients with recurrent human glioma [167Rampling R, Cruickshank G, Papanastassiou V, et al. Toxicity evaluation of replication-competent herpes simplex virus (ICP 34.5 null mutant 1716) in patients with recurrent malignant glioma Gene Ther 2000; 7: 859-66.-169Harrow S, Papanastassiou V, Harland J, et al. HSV1716 injection into the brain adjacent to tumour following surgical resection of high-grade glioma: safety data and long-term survival Gene Ther 2004; 11: 1648-58.], after it was demonstrated to be avirulent in mice. HSV1716 has also been injected into subcutaneous nodules of metastatic melanoma in five patients and local evidence of anti-tumour effects was observed [170Randazzo BP, Kesari S, Gesser RM, et al. Treatment of experimental intracranial murine melanoma with a neuroattenuated herpes simplex virus 1 mutant Virology 1995; 211: 94-101.]. HSV1716 will move soon in phase I clinical trial for non-CNS solid tumours.

Oncolytic herpesvirus have been also studied as an oncolytic anti-tumour therapy against a variety of tumours different than GBM and anaplastic astrocytoma, such as human breast cancer in a brain metastatic model [171Toda M, Rabkin SD, Martuza RL. Treatment of human breast cancer in a brain metastatic model by G207, a replication-competent multimutated herpes simplex virus 1 Hum Gene Ther 1998; 9: 2177-85.], colorectal cancer and liver metastases [172Kooby DA, Carew JF, Halterman MW, et al. Oncolytic viral therapy for human colorectal cancer and liver metastases using a multi-mutated herpes simplex virus type-1 (G207) FASEB J 1999; 13: 1325-34.], prostate cancer [173Cozzi PJ, Burke PB, Bhargav A, et al. Oncolytic viral gene therapy for prostate cancer using two attenuated, replication-competent, genetically engineered herpes simplex viruses Prostate 2002; 53: 95-100.], pancreatic cancer [174McAuliffe PF, Jarnagin WR, Johnson P, et al. Effective treatment of pancreatic tumors with two multimutated herpes simplex oncolytic viruses J Gastrointest Surg 2000; 4: 580-8.], and head and neck squamous carcinoma [175Carew JF, Kooby DA, Halterman MW, Federoff HJ, Fong Y. Selective infection and cytolysis of human head and neck squamous cell carcinoma with sparing of normal mucosa by a cytotoxic herpes simplex virus type 1 (G207) Hum Gene Ther 1999; 10: 1599-606.]. In many of these studies, the efficacy of G207 has been compared with that of the HSV-1 mutant NV1020 (formerly R7020) strain [121Meignier B, Longnecker R, Roizman B. In vivo behavior of genetically engineered herpes simplex viruses R7017 and R7020: construction and evaluation in rodents J Infect Dis 1988; 158: 602-14., 122Meignier B, Martin B, Whitley RJ, Roizman B. In vivo behavior of genetically engineered herpes simplex viruses R7017 and R7020. II. Studies in immunocompetent and immunosuppressed owl monkeys (Aotus trivirgatus) J Infect Dis 1990; 162: 313-21.], which was the first attenuated HSV virus to be constructed and analyzed as a viral vaccine in humans. This virus, based on HSV-1 strain F, has a portion of the unique short region of the viral genome, encoding glycoproteins G, D, I and E, replaced by the homologous region from HSV-2, and possesses one copy of ICP4. NV1020, which was shown to be too attenuated to work as a live viral vaccine, is currently being investigated in phase I and II clinical trials for patients with colon cancer that has metastasized to the liver and has proven recalcitrant to chemotherapy [176Kelly KJ, Wong J, Fong Y. Herpes simplex virus NV1020 as a novel and promising therapy for hepatic malignancy Exp Opin Investig Drugs 2008; 17: 1105-3., 177Fong Y, Kim T, Bhargava A, et al. A herpes oncolytic virus can be delivered via the vasculature to produce biologic changes in human colorectal cancer Mol Ther 2009; 17: 389-94.]. This is also the first trial to investigate administration via intravascular delivery. In fact, oncolytic viruses can be administered locally, by direct intratumoral inoculation, or systemically, by intravascular administration. However, the route of administration of the virus did influence efficacy, as was observed in the animal model. The results demonstrated that NV1020 is a safe, novel therapeutic agent for treatment of refractory hepatic malignacy. An evolution of NV1020, namely NV1023, in which it has been inserted an HSV-2 fragment containing genes US2-2 and US2-5 into the UL/S junction and other minor mutations, is under evaluation, combined with ionizing radiation, in human cholangiocarcinoma cell lines and could also be useful for other malignancies such as pancreatic, rectal, prostate, and head/neck cancer [178Jarnagin WR, Zager JS, Hezel M, et al. Treatment of cholangiocarcinoma with oncolytic herpes simplex virus combined with external beam radiation therapy Cancer Gene Ther 2006; 13: 326-4.]. Finally, it has been recently demonstrated that highly fusogenic derivatives of NV1020, named OncSyn and OncdSyn, carrying syncytial mutations in both gB (syn3) (OncSyn) and in gB and gK (syn1) (OncdSyn), can reduce primary and metastatic breast tumours in immunocompetent mice [179Israyelyan AH, Melancon JM, Lomax LG, et al. Effective treatment of human breast tumor in a mouse xenograft model with herpes simplex virus type 1 specifying the NV1020 genomic deletion and the gBsyn3 syncytial mutation enabling high viral replication and spread in breast cancer cells Hum Gene Ther 2007; 18: 457-73.-181Israyelyan A, Chouljenko VN, Baghian A, et al. Herpes simplex virus type-1(HSV-1) oncolytic and highly fusogenic mutants carrying the NV1020 genomic deletion effectively inhibit primary and metastatic tumors in mice Virol J 2008; 5: 68.].

G47Δ is a third-generation vector that was constructed by deletion of US12 gene, encoding ICP47 protein, which normally blocks MHC class I-mediated antigen presentation in infected cells. Consequently, human melanoma cells infected with G47Δ expressed higher levels of MHC class I on their surface, compared to G207-infected cells, resulting in enhanced stimulation of tumour-infiltrating lymphocytes. The US12 deletion also removes the US11 promoter, so that US11 gene is expressed as an immediate-early gene under the control of the US12 promoter, thereby suppressing the diminished growth properties of ICP34.5 mutants [144Todo T, Martuza RL, Rabkin SD, Johnson PA. Oncolytic herpes simplex virus vector with enhanced MHC class I presentation and tumor cell killing Proc Natl Acad Sci USA 2001; 98: 6396-401.]. This improved replication of G47Δ translates into enhanced antitumor activity. Another HSV vector with the same characteristics of G47Δ was isolated after serial passages, of an ICP34.5 deleted HSV, on a tumour cell lines. This viral isolate was shown to exhibit enhanced antitumor effect on prostate cancer [182Taneja S, MacGregor J, Markus S, Ha S, Mohr I. Enhanced antitumor efficacy of a herpes simplex virus mutant isolated by genetic selection in cancer cells Proc Natl Acad Sci USA 2001; 98: 8804-.].

Despite the promising results obtained with the engineered HSV-1-based oncolytic vectors described above, it is likely that a multimodal approach to eradicate cancer will be more effective with the final goal to improve safety and efficacy of the system. At this regard, oncolytic HSV vectors have been further modified to augment their antitumor efficacy, by incorporation of expression cassettes for the delivery of various transgenes. Moreover, if the therapeutic gene is chosen carefully, this may be synergistic with the antitumor effect of virus replication.

In early clinical trials, however, treatment with the current generation of oncolytic viruses did not significantly affect tumour growth [163Markert JM, Medlock MD, Rabkin SD, et al. Conditionally replicating herpes simplex virus mutant, G207 for the treatment of malignant glioma: results of a phase I trial Gene Ther 2000; 7: 867-74., 167Rampling R, Cruickshank G, Papanastassiou V, et al. Toxicity evaluation of replication-competent herpes simplex virus (ICP 34.5 null mutant 1716) in patients with recurrent malignant glioma Gene Ther 2000; 7: 859-66.]. This suboptimal result may reflect viral gene deletions, which can reduce the replicative potential of viruses in tumour cells. For example, deletion of the γ-34.5 gene significantly reduced viral growth even in rapidly dividing cells [144Todo T, Martuza RL, Rabkin SD, Johnson PA. Oncolytic herpes simplex virus vector with enhanced MHC class I presentation and tumor cell killing Proc Natl Acad Sci USA 2001; 98: 6396-401., 149Kramm CM, Rainov NG, Sena-Esteves M, et al. Long-term survival in a rodent model of disseminated brain tumors by combined intrathecal delivery of herpes vectors and ganciclovir treatment Hum Gene Ther 1996; 7: 1989-94., 191Andreansky S, He B, van Cott J, et al. Treatment of intracranial gliomas in immunocompetent mice using herpes simplex viruses that express murine interleukins Gene Ther 1998; 5: 121-30.]. A variety of strategies is being pursued to enhance the potency of oncolytic viruses by “arming” them with various transgenes. Overall, the results so far obtained demonstrate that this last generation HSV vectors can increase antitumor efficacy, especially if used in combination with pre-existing anticancer treatments, such as radiotherapy or chemotherapy [18Post DE, Fulci G, Chiocca EA, Van Meir EG. Replicative oncolytic herpes simplex viruses in combination cancer therapies Curr Gene Ther 2004; 4: 41-51., 186Aghi M, Chou TC, Suling K, Breakefield XO, Chiocca EA. Multimodal cancer treatment mediated by a replicating oncolytic virus that delivers the oxazaphosphorine/rat cytochrome P450 2B1 and ganciclovir/herpes simplex virus thymidine kinase gene therapies Cancer Res 1999; 59: 3861-5., 226He B, Chou J, Brandimarti R, et al. Suppression of the phenotype of gamma(1)34.5- herpes simplex virus 1: failure of activated RNA-dependent protein kinase to shut off protein synthesis is associated with a deletion in the domain of the alpha47 gene J Virol 1997; 71: 6049-54., 227Jorgensen TJ, Katz S, Wittmack EK, et al. Ionizing radiation does not alter the antitumor activity of herpes simplex virus vector G207 in subcutaneous tumor models of human and murine prostate cancer Neoplasia 2001; 3: 451-6.]. Combined oncolytic viral therapy and external beam radiotherapy (XRT) has shown significantly improved results over individual therapies in preclinical models [228Ottolino-Perry K, Diallo JS, Lichty BD, Bell JC, Andrea McCart J. Intelligent design: combination therapy with oncolytic viruses Mol Ther 2010; 18: 251-63.]. Significant improvements in disease outcomes have been observed with combination HSV virotherapy and XRT in preclinical models. In two different studies, NV1066 (ICP0/ICP4/γ34.5 deletions) combined with irradiation was shown to significantly reduce tumour volume compared to either treatment alone for nonsmall cell lung cancer and malignant mesothelioma [229Blank SV, Rubin SC, Coukos G, et al. Replication-selective herpes simplex virus type 1 mutant therapy of cervical cancer is enhanced by low-dose radiation Hum Gene Ther 2002; 13: 627-39., 230Adusumilli PS, Chan MK, Hezel M, et al. Radiation-induced cellular DNA damage repair response enhances viral gene therapy efficacy in the treatment of malignant pleural mesothelioma Ann Surg Oncol 2007; 14: 258-69.]. Moreover, the combination of NV1023 (γ34.5/UL24/UL56/US11/ICP47 deletions) with XRT was investigated in three models of cholangiocarcinoma generated using different cell lines [178Jarnagin WR, Zager JS, Hezel M, et al. Treatment of cholangiocarcinoma with oncolytic herpes simplex virus combined with external beam radiation therapy Cancer Gene Ther 2006; 13: 326-4.]. This dual therapy showed a synergistic reduction in tumour volume in one model, whereas the effect was additive or not significantly better than individual therapies in the other models. Despite the promising results described above, the potential for combination therapy remains to be seen in a variety of other cancers. For example, in one study the combination of HSV oncolytic therapy and XRT, in prostate models, was not significantly better than virus therapy alone in both immunocompetent and immunoco-mpromised models [227Jorgensen TJ, Katz S, Wittmack EK, et al. Ionizing radiation does not alter the antitumor activity of herpes simplex virus vector G207 in subcutaneous tumor models of human and murine prostate cancer Neoplasia 2001; 3: 451-6.]. The underlying mechanism of increased HSV replication and oncolytic effect in the presence of XRT has been well characterized studying the synergy between ICP34.5-deleted viruses and conventional cancer therapy [231Advani SJ, Mezhir JJ, Roizman B, Weichselbaum RR. ReVOLT: radiation-enhanced viral oncolytic therapy Int J Radiat Oncol Biol Phys 2006; 66: 637-46.]. Interestingly, ICP34.5 shows significantly structural homology to a portion of human GADD34, a protein involved in the cell response to DNA damage [232Chou J, Roizman B. Herpes simplex virus 1 gamma(1)34.5 gene function, which blocks the host response to infection, maps in the homologous domain of the genes expressed during growth arrest and DNA damage Proc Natl Acad Sci USA 1994; 91: 5247-1.]. ICP34.5 is responsible for dephosphorilation of eIF-2α, which is required for translation of both host and viral proteins. Radiation or chemotherapy-induced up regulation of GADD34 functionally replaces the ICP34.5 protein in infected tumour cells leading to increase viral protein synthesis and production of infectious virus particles.

Oncolytic HSVs have been also investigated in combination with various standard chemotherapeutics, such as cyclophosphamide (CPA) and cisplatin. Use of CPA as an immunosuppressant to enhance viral oncolysis has improved virotherapy efficacy in combination with HSV [233Ikeda K, Wakimoto H, Ichikawa T, et al. Complement depletion facilitates the infection of multiple brain tumors by an intravascular, replication-conditional herpes simplex virus mutant J Virol 2000; 74: 4765-75.-235Fulci G, Breymann L, Gianni D, et al. Cyclophosphamide enhances glioma virotherapy by inhibiting innate immune responses Proc Natl Acad Sci USA 2006; 103: 12873-8.]. However, one of the potential pitfalls of CPA-mediated immune suppression is that in addition to promoting tumour oncolysis, it may also lead to increased virus dissemination through the body. In this regard, it has been observed that CPA can induce the spread of HSV into normal brain tissue following treatment of an orthotopic glioma tumour [236Wakimoto H, Fulci G, Tyminski E, Chiocca EA. Altered expression of antiviral cytokine mRNAs associated with cyclophosphamide's enhancement of viral oncolysis Gene Ther 2004; 11: 214-3.]. Cisplatin has also been investigated in combination with oncolytic HSV [237Adusumilli PS, Chan MK, Chun YS, et al. Cisplatin-induced GADD34 upregulation potentiates oncolytic viral therapy in the treatment of malignant pleural mesothelioma Cancer Biol Ther 2006; 5: 48-53.-239Mace AT, Harrow SJ, Ganly I, Brown SM. Cytotoxic effects of the oncolytic herpes simplex virus HSV1716 alone and in combination with cisplatin in head and neck squamous cell carcinoma Acta Otolaryngol 2007; 127: 880-7.]. In a comprehensive study of the interaction between NV1066 HSV vector and cisplatin, moderate to strong synergy was observed. The mechanism of synergy may be similar to that with radiation. Cisplatin significantly increased viral titer and resulted in a marked increase in GADD34 mRNA and protein expression.

HSV induce both innate and adaptive immunity in the infected host. The principal component of innate immunity that control HSV infection are: macrophages [282Ellermann-Eriksen S. Macrophages and cytokines in the early defence against herpes simplex virus Virol J 2005; 2: 59-0.], polymorphonuclear leukocytes (PMNLs), natural killer (NK) cells and innate NK-like T cells (iNKT) [283Nandakumar S, Woolard SN, Yuan D, Rouse BT, Kumaraguru U. Natural killer cells as novel helpers in anti-herpes simplex virus immune response J Virol 2008; 82: 10820-31., 284Diana J, Lehuen A. NKT cells: Friend or foe during viral infections? Eur J Immunol 2009; 39: 3283-91.], and dendritic cells (DCs) [282Ellermann-Eriksen S. Macrophages and cytokines in the early defence against herpes simplex virus Virol J 2005; 2: 59-0.]. Dendritic cells have a crucial role and function as a bridge between innate and adaptive immunity. Adaptive response to HSV infection include the cellular response mediated by CD4⁺ and CD8⁺ T cells and the humoral response by B cells and antibodies.

Chemokines [285Carr DJ, Tomanek L. Herpes simplex virus and the chemokines that mediate the inflammation Curr Top Microbiol Immunol 2006; 303: 47-65., 286Wuest TR, Carr DJ. The role of chemokines during herpes simplex virus-1 infection Front Biosci 2008; 13: 4862-72.] and cytokines [279Broberg EK, Hukkanen V. Immune response to herpes simplex virus and gamma134.5 deleted HSV vectors Curr Gene Ther 2005; 5: 523-30., 287Hukkanen V, Broberg E, Salmi A, Eralinna JP. Cytokines in experimental herpes simplex virus infection Int Rev Immunol 2002; 21: 355-71.] have an important role in the coordination and development of innate and adaptive immune response to HSV-1. Control of HSV-1 during primary infection requires recruitment of PMNLs macrophages and NK cells by specific chemokines, and culminates in the induction of antigen specific responses by T cells which control viral replication through lysis of infected cells and cytokine production. Effective innate control of viral replication may lower latent viral burden present in sensory ganglia, and reduce the frequency of subsequent reactivation. Cytokine production at the site of infection and in the sensory ganglion has been reported to affect the course of HSV infection. Several cytokines have been detected in these tissue during HSV infection both in human and in experimental animals. The cytokines detected include IFN-α, -β and -γ, IL-1, -2, -4, -5, -6, -10, -12 and -23 and also TNF-α [279Broberg EK, Hukkanen V. Immune response to herpes simplex virus and gamma134.5 deleted HSV vectors Curr Gene Ther 2005; 5: 523-30., 287Hukkanen V, Broberg E, Salmi A, Eralinna JP. Cytokines in experimental herpes simplex virus infection Int Rev Immunol 2002; 21: 355-71.]. IFN-α and -β limit the early acute replication of HSV [288Leib DA, Harrison TE, Laslo KM, et al. Interferons regulate the phenotype of wild-type and mutant herpes simplex viruses in vivo J Exp Med 1999; 189: 663-72.]. IFN-γ production and the presence of CD4⁺ and CD8⁺ T cells are key regulators of viral clearance during acute infection. Other proinflammatory cytokines are involved in regulation of viral replication and reactivation in sensory ganglia, such as IL-1, -6, -12 and TNF-α. IL-12 has important roles in the development of Th1 response and in cell-mediated immunity. It has been reported that IL-23 is expressed in the brain during infection [289Broberg EK, Setala N, Eralinna JP, et al. Herpes simplex virus type 1 infection induces upregulation of interleukin-23 (p19) mRNA expression in trigeminal ganglia of BALB/c mice J Interferon Cytokine Res 2002; 22: 641-51.]. It is suggested a major role for IL-23 in the proinflammatory response [287Hukkanen V, Broberg E, Salmi A, Eralinna JP. Cytokines in experimental herpes simplex virus infection Int Rev Immunol 2002; 21: 355-71., 289Broberg EK, Setala N, Eralinna JP, et al. Herpes simplex virus type 1 infection induces upregulation of interleukin-23 (p19) mRNA expression in trigeminal ganglia of BALB/c mice J Interferon Cytokine Res 2002; 22: 641-51., 290Wiekowski MT, Leach MW, Evans EW, et al. Ubiquitous transgenic expression of the IL-23 subunit p19 induces multiorgan inflammation, runting, infertility, and premature death J Immunol 2001; 166: 7563-0.]. The role of Th2 type cytokines in HSV infection has not been well established. IL-4 and IL-10 expression has been detected early during the infection in sensory ganglia of HSV-susceptible mouse strains [291Shimeld C, Easty DL, Hill TJ. Reactivation of herpes simplex virus type 1 in the mouse trigeminal ganglion: an in vivo study of virus antigen and cytokines J Virol 1999; 73: 1767-73.].

HSV1-based vector are particularly amenable to gene therapy applications within the CNS. Because aspects of the innate immune response to HSV-1 vectors in CNS are largely unknown, it is important to develop new tools for such studies. In this regards, Zeier and colleagues [329Zeier Z, Aguilar JS, Lopez CM, et al. A limited innate immune response is induced by a replication-defective herpes simplex virus vector following delivery to the murine central nervous system J Neurovirol 2009; 15: 411-24.] have recently compared the host response of a replication-defective HSV-1 vector to that of a replication-competent HSV-1 using microarray analysis. Pathway analysis revealed that both the replicating and nonreplicating vectors induced robust antigen presentation but only mild interferon, chemokine, and cytokine signalling responses. The ICP4 deleted replication-defective vector was restricted in several of the TLR receptor-signalling pathways, indicating reduced stimulation of innate immune response. These array analyses suggest that although the replication-defective vector induces detectable activation of immune response pathway, the number and magnitude of the induced response is dramatically restricted compared to the replicating vector and with the exception of antigen presentation, host gene expression induced by nonreplicating vector resembles mock infection.

Innate immunity is critically important in limiting wt viral infections and in the context of HSV oncolytic therapy, this branch of the immune system appears to be a potent obstacle for achieving oncolytic vector replication and tumour destruction [330Haseley A, Alvarez-Breckenridge C, Chaudhury AR, Kaur B. Advances in oncolytic virus therapy for glioma Recent Pat CNS Drug Discov 2009; 4: 1-13.]. In this regard, drugs that inhibit innate immune response have been shown to enhance glioma virotherapy [235Fulci G, Breymann L, Gianni D, et al. Cyclophosphamide enhances glioma virotherapy by inhibiting innate immune responses Proc Natl Acad Sci USA 2006; 103: 12873-8.]. Moreover, both the antitumor efficacies as well as the intratumoral viral titers were found to be significantly increased with the concurrent depletion of mononuclear cells and the elimination of antiviral cytokines [235Fulci G, Breymann L, Gianni D, et al. Cyclophosphamide enhances glioma virotherapy by inhibiting innate immune responses Proc Natl Acad Sci USA 2006; 103: 12873-8.].

Following activation of innate immunity, the adaptive component of the immune system is recruited to the site of infection and participates in both the killing of virally infected cells and the production of antibodies against foreign antigens. The induction of an immune response against antiviral products can represent the major problem and may reduce the effectiveness of HSV-1-derived vectors. In fact, immune responses arising from the delivery of recombinant HSV vectors can be due to several factors, such as: viral particles components copurified packaging cell debris, different routes of delivery, multiple injections of the vectors, low-level de novo viral gene product expression. As a consequence of this last aspect, replication-defective vectors may contains ORFs that are expressed at low levels even in the absence of immediate-early gene products, augmenting the potential for antigen processing and subsequent MHC class I presentation.

HSV-based vectors have been used in a variety of diverse applications including gene transfer for gene therapy, as oncolytic viruses, or as a vaccination platform. In the case of gene transfer, an immune response against the vector or the transgene would be detrimental to sustained transgene expression, weakening the efficiency of the therapy. Targeted oncolytic viruses must replicate in and destroy cancer cells selectively enhancing, at the same time, tumour-specific immunity. Alternatively, when using HSV as a vaccination platform the preferred outcome is a directed immune response to the transgene. The desired results of different applications in gene therapy might be opposites and would require different strategies in constructing HSV-based vectors.

To prolong the effect of transgenes expressed from viral vectors in gene transfer therapy, it has been shown that treatment with cyclosporine A can reduce the inflammatory response to a HSV vector leading to improved transgene expression [331Mabon PJ, Weaver LC, Dekaban GA. Cyclosporin A reduces the inflammatory response to a multi-mutant herpes simplex virus type-1 leading to improved transgene expression in sympathetic preganglionic neurons in hamsters J Neurovirol 1999; 5: 268-79.], while immunomodulative treatment with Linomide seems to facilitates the spread and expression of ICP34.5-negative vector in CNS [332Peltoniemi J, Broberg EK, Nygardas M, et al. Enhancement of Th2 responses to replicative herpes simplex virus type 1 vectors by immunomodulative chemotherapy Int Immunopharmacol 2006; 6: 817-29.].

In oncolytic virotherapy, CTLs have been implicated as the critical responders to viral antigens presented on the surface of tumour cells. CTLs are subsequently redirected to tumour cell antigens, thereby enhancing the efficacy of oncolytic HSV-1 by inducing antitumor immunity [144Todo T, Martuza RL, Rabkin SD, Johnson PA. Oncolytic herpes simplex virus vector with enhanced MHC class I presentation and tumor cell killing Proc Natl Acad Sci USA 2001; 98: 6396-401., 333Thomas DL, Fraser NW. HSV-1 therapy of primary tumors reduces the number of metastases in an immune-competent model of metastatic breast cancer Mol Ther 2003; 8: 543-1.]. In this regard, the inclusion of genes encoding for various cytokines into viral vectors to enhance antitumor immune responses has been also tested and found to be beneficial in several preclinical models of cancer [204Hu JC, Coffin RS, Davis CJ, et al. A phase I study of OncoVEXGM-CSF, a second-generation oncolytic herpes simplex virus expressing granulocyte macrophage colony-stimulating factor Clin Cancer Res 2006; 12: 6737-47., 206Varghese S, Rabkin SD, Liu R, et al. Enhanced therapeutic efficacy of IL-12, but not GM-CSF, expressing oncolytic herpes simplex virus for transgenic mouse derived prostate cancers Cancer Gene Ther 2006; 13: 253-65.]. As a further improvement in oncolytic virotherapy, it has been observed that apoptotic bodies produced upon viral infection and their subsequent engulfment by APCs can potentiate cytotoxic T lymphocyte activity and the anti-tumour response. The potential to exploit this in conjunction with OV therapy to enhance anti-tumour immunity is only beginning to be realized and needs to be further investigated [330Haseley A, Alvarez-Breckenridge C, Chaudhury AR, Kaur B. Advances in oncolytic virus therapy for glioma Recent Pat CNS Drug Discov 2009; 4: 1-13.].

ICP34.5-deleted HSV vectors have been extensively used both in gene transfer therapy and in oncolytic virotherapy. However, immune response to these vectors has not been thoroughly elucidated. It has shown that infection with ICP34.5-deleted HSV vectors results in alterations in splenocyte subsets and cytokine expression [334Broberg EK, Salmi AA, Hukkanen V. IL-4 is the key regulator in herpes simplex virus-based gene therapy of BALB/c experimental autoimmune encephalomyelitis Neurosci Lett 2004; 364: 173-8.]. The viruses carrying IL-4 or IL-10 transgenes or even the ICP34.5-deleted HSV vectors without transgenes have been shown to induce Th2 type cytokine response, whereas the wt HSV-1 induces Th1 type cytokine response in immunological cell populations. However, in the CNS the cytokine expression was more variable [334Broberg EK, Salmi AA, Hukkanen V. IL-4 is the key regulator in herpes simplex virus-based gene therapy of BALB/c experimental autoimmune encephalomyelitis Neurosci Lett 2004; 364: 173-8.]. Moreover, it has been reported that virus R8306, expressing IL-4 transgene suppressed IFN-γ and induced IL-23 expression during the acute infection but decreased its expression during the later time points. Furthermore, R8308 virus without transgenes or R8308 with IL-10 transgene induced IFN-γ as well as IL-23 responses in the brain. The virus R3616 with the deletion of both ICP34.5 genes causes differences in the splenocyte subsets in comparison to the wild-type infection, inducing a stronger proliferation of CD4+ and CD8+T cells, as well as of the CD11c+ antigen presenting cells [334Broberg EK, Salmi AA, Hukkanen V. IL-4 is the key regulator in herpes simplex virus-based gene therapy of BALB/c experimental autoimmune encephalomyelitis Neurosci Lett 2004; 364: 173-8.]. This indicate an additional role of ICP34.5 as to those already observed, such as immune evasion and inhibition of the host interferon response [335Cheng G, Yang K, He B. Dephosphorylation of eIF-2alpha mediated by the gamma(1)34.5 protein of herpes simplex virus type 1 is required for viral response to interferon but is not sufficient for efficient viral replication J Virol 2003; 77: 10154-61., 336Cheng G, Brett ME, He B. Val193 and Phe195 of the gamma 1 34.5 protein of herpes simplex virus 1 are required for viral resistance to interferon-alpha/beta Virology 2001; 290: 115-20.]. The immune responses evoked by the ICP34.5-deleted vectors used in the clinical cancer therapies (G207, 1716, NV1020 and OncoVex) have not been reported and can therefore not yet be compared with the reported results. The immunological reactions due to the HSV vector are needed to be studied in more detail. The specific Th1 or Th2 type cytokine responses can be used to benefit the therapy. The ICP34.5-deleted HSV vectors are excellent basis and platform for the development of even more attenuated and safe vectors, such as to avoid some of the risk factors described above.

Concerning the use of viral vector as vaccines, numerous strategies have attempted to influence or direct the immune response toward either a Th1 type or Th2 type; the presence of particular cytokines is one way to accomplish this goal. The presence of IFN-γ and IL-1 will push the development of a Th1-type response. This type of response is useful when cellular immunity and inflammation are desired. The presence of IL-4 and IL-10 will skew a response toward the Th2 type. These cytokines bring about a strong humoral response, avoiding the often unnecessary, and dangerous, inflammation and presence of CTLs. The preferred cytokine gene can be inserted into the HSV-based vector, allowing for its coexpression with the antigen of interest, increasing the likelihood that a particular response is established. Another consideration when attempting to direct an adaptive response is the choice of transgene within the vector, since whole gene products may contain several different epitopes, which can be T or B cell epitopes. Expressing only a portion of an antigen of interest, including a specific epitope, or coexpressing an epitope that is a strong inducer of a known response type, can push the response toward one that is more humoral or cellular in nature.

It has been recently demonstrated that degradation of the fibrillar collagen network by collagenase, in a human melanoma xenograft, favours the regression of tumour vasculature and improves the distribution and the efficacy of oncolytic HSV in tumour tissue [343McKee TD, Grandi P, Mok W, et al. Degradation of fibrillar collagen in a human melanoma xenograft improves the efficacy of an oncolytic herpes simplex virus vector 10.1158/0008-5472.CAN- 05-2242 Cancer Res 2006; 66: 2509-13.]. Successively, it has been shown that human matrix metalloproteinases-1 and -8 (MMP-1 or MMP-8), which degrade fibrillar collagens, proteoglycans, and other structural matrix components, enhance efficacy of oncolytic HSV therapy [344Mok W, Boucher Y, Jain RK. Matrix metalloproteinases-1 and -8 improve the distribution and efficacy of an oncolytic virus Cancer Res 2007; 67: 10664-8.]. Therefore, intratumoral expression of MMPs seems to be a promising strategy for improving viral spread and therapeutic potential of oncolytic viruses by modulation of tumour ECM.

However, some concerns about strategies that cause degradation of ECM exist. While degradation of ECM with enzymes, such as collagenase and MMPs, may improve viral penetration and distribution, on the other hand may also increase tumour spread. In fact, MMPs and collagenase have been shown to play an important role in tumour invasion and metastasis [345Deryugina EI, Quigley JP. Matrix metalloproteinases and tumor metastasis Cancer Metastasis Rev 2006; 25: 9-34.]. Even if, there is also some emerging evidence that MMP-8 does not promote tumorigenesis or metastasis and may be antimetastatic [346Montel V, Kleeman J, Agarwal D, et al. Altered metastatic behavior of human breast cancer cells after experimental manipulation of matrix metalloproteinase 8 gene expression Cancer Res 2004; 64: 1687-94.]. Therefore, further thorough and detailed studies are required to gain an improved understanding of the potential risk associated with combined replicating oncolytic virus and ECM-degrading enzyme.

Induction of apoptosis in the tumour mass can facilitate the release and distribution of viruses. Interestingly, apoptosis induced during viral DNA replication seems to compromise viral production, whereas apoptosis induced after virion assembly enhances viral release and dissemination from infected cells [297Nguyen ML, Blaho JA. Apoptosis during herpes simplex virus infection Adv Virus Res 2007; 69: 67-97., 347Huszthy PC, Immervoll H, Wang J, et al. Cellular effects of oncolytic viral therapy on the glioblastoma microenvironment Gene Ther 2010; 17: 202-16.]. Apoptosis is significantly elevated in tumour tissue treated with oncolytic viruses and chemotherapeutics, as chemotherapy-induced cell death occurs mainly by the apoptotic pathway. Therefore, tumour cell apoptosis could be at the basis of the strong synergy observed between oncolytic HSV with various chemotherapeutics [348Watanabe I, Kasuya H, Nomura N, et al. Effects of tumor selective replication-competent herpes viruses in combination with gemcitabine on pancreatic cancer Cancer Chemother Pharmacol 2008; 61: 875-2.]. As it has been described above, interstitial collagen fibers and the narrow spacing (~ 20 nm) between cancer cells are major barriers hindering the movement of large viral particles and limit the success of oncolytic virotherapy. The void space produced by cancer cell apoptosis might enhance the initial spread and efficacy of oncolytic HSV. At this regard, it has been reported that Paclitaxel plus TRAIL pretreatment, two inducing agents of apoptosis, enhances oncolytic HSV intratumoral delivery/ penetration and antitumor efficacy [349Nagano S, Perentes JY, Jain RK, Boucher Y. Cancer cell death enhances the penetration and efficacy of oncolytic herpes simplex virus in tumors Cancer Res 2008; 68: 3795-802.]. These observations emphasize the important relationship between induction of the apoptotic pathway and viral spread in solid-tumour tissue. The formation of apoptotic bodies, a morphological characteristic of apoptosis, can also facilitate the dispersal of viral particles throughout the tumour mass. Apoptotic bodies can also be rapidly phagocytosed by neighbouring cells, such as macrophages and tumour cells, and disperse the virus throughout the intercellular spaces. Viral dissemination through apoptotic bodies may be one of the major contributors for the synergistic therapeutic effect of oncolytic virotherapy and chemotherapy.

Viral spread within the tumour mass cannot be investigated using conventional monolayer cell cultures, because these models poorly mimic the solid tumour microenvironment. An alternative is multicellular tumour spheroids (MTSs), which have gained in importance as an in vitro model of solid tumours [350Lin RZ, Chang HY. Recent advances in three-dimensional multicellular spheroid culture for biomedical research Biotechnol J 2008; 3: 1172-84.]. The 3-dimensional (D) MTSs are intermediate in complexity between standard 2-D monolayer cultures in vitro and tumour tissues in vivo. They consist entirely of tumour cells and therefore fall to represent the native 3-D architecture and heterogeneity of solid tumours in vivo. As an example, glioblastoma (GBM) biopsy material obtained at surgery, has been cultured in the form of multicellular spheroids, where the essential phenotypic characteristics of the tumour tissue are retained [351Bjerkvig R, Tonnesen A, Laerum OD, Backlund EO. Multicellular tumor spheroids from human gliomas maintained in organ culture J Neurosurg 1990; 72: 463-75.]. Direct transplantation of such “organotypic” spheroids into rodent brains forms lesions that eventually recapitulate all the histopathological infiltration, angiogenesis, endothelial proliferations and dilated, thrombotic vessels [352Engebraaten O, Hjortland GO, Hirschberg H, Fodstad O. Growth of precultured human glioma specimens in nude rat brain J Neurosurg 1999; 90: 125-32.]. The effect of oncolytic G207 virus, currently used in clinical therapy, has been assessed on a set of xenograft phenotypes that follow these biological features described above [347Huszthy PC, Immervoll H, Wang J, et al. Cellular effects of oncolytic viral therapy on the glioblastoma microenvironment Gene Ther 2010; 17: 202-16.]. The study highlights the favourable cellular responses to G207 treatment seen from a clinical viewpoint, such as reduced tumour cell proliferation, more frequent events of tumour cell death, and a strongly attenuated tumour vascular compartment. However, these beneficial changes were only observed in areas of active viral replication, leaving non-transduced tumour tissues unaffected.