Effects of herpes simplex virus vectors encoding poreless TRPV1 or protein phosphatase 1α in a rat cystitis model induced by hydrogen peroxide.

Enhanced afferent excitability is considered to be an important pathophysiological basis of interstitial cystitis/bladder pain syndrome (IC/BPS). In addition, transient receptor potential vanilloid-1 (TRPV1) receptors are known to be involved in afferent sensitization. Animals with hydrogen peroxide (HP)-induced cystitis have been used as a model exhibiting pathologic characteristics of chronic inflammatory condition of the bladder. This study investigated the effect of gene therapy with replication-defective herpes simplex virus (HSV) vectors encoding poreless TRPV1 (PL) or protein phosphatase 1 α (PP1α), a negative regulator of TRPV1, using a HP-induced rat model of cystitis. HSV vectors encoding green fluorescent protein, PL or PP1α were inoculated into the bladder wall of female rats. After 1 week, 1% HP or normal saline was administered into the bladder, and the evaluations were performed 2 weeks after viral inoculation. In HP-induced cystitis rats, gene delivery of PL or PP1α decreased pain behavior as well as a reduction in the intercontraction interval. Also, both treatments reduced nerve growth factor expression in the bladder mucosa, reduced bladder inflammation characterized by infiltration of inflammatory cells and increased bladder weight. Taken together, HSV-mediated gene therapy targeting TRPV1 receptors could be effective for the treatment of IC/BPS.

MnSOD mediated by HSV vectors in the periaqueductal gray suppresses morphine withdrawal in rats.

Morphine appears to be the most active metabolite of heroin; therefore, the effects of morphine are important in understanding the ramifications of heroin abuse. Opioid physical dependence (withdrawal response) may have very long-lasting effects on the motivation for reward, including the incubation of cue-induced drug-seeking behavior. However, the exact mechanisms of morphine withdrawal (MW) are not clear yet, and its treatment remains elusive. Periaqueductal gray (PAG) is one of the important sites in the pathogenesis of MW. Here, we used recombinant herpes simplex virus (HSV) vectors that encode the sod2 gene expressing manganese superoxide dismutase (MnSOD) to evaluate its therapeutic potential in MW. Microinjection of HSV vectors expressing MnSOD into the PAG reduced the MW syndrome. MnSOD vectors suppressed the upregulated mitochondrial superoxide, and endoplasmic reticulum stress markers (glucose-related protein 78 (GRP78) and activating transcription factor 6 alpha (ATF6α)) in the PAG induced by MW. Immunostaining showed that mitochondrial superoxide, GRP78 and ATF6α were colocalized with neuronal nuclei (a neuronal-specific marker), suggesting that they are located in the neurons in the PAG. These results suggest that overexpression of MnSOD by HSV vectors may relieve opioid dependence. This study may provide a novel therapeutic approach to morphine physical withdrawal response.

Development of an oncolytic HSV vector fully retargeted specifically to cellular EpCAM for virus entry and cell-to-cell spread.

Oncolytic herpes simplex virus (HSV) vectors have attracted increasing attention as novel anti-cancer agents. HSV entry is triggered by the binding of glycoprotein D (gD) to its receptors, such as herpesvirus entry mediator or nectin-1. We have recently reported the construction of a fully retargeted HSV platform that incorporates single-chain antibodies (scFv) into gD to mediate entry exclusively via tumor-associated antigens. In this study, we created an scFv directed against epithelial cell adhesion molecule (EpCAM), a recognized carcinoma-associated antigen, and inserted it into the retargeted HSV platform that is ablated for gD recognition of its canonical receptors and contains the entry-enhancing mutations in gB we previously identified. We observed that both initial entry and subsequent cell-to-cell spread of the retargeted virus were stringently dependent on cellular EpCAM expression. Interestingly, the retargeted virus developed larger plaques on some of the human tumor lines tested than the control virus bearing wild-type gD. Intratumoral injection of the retargeted virus revealed antitumor activity in a mouse xenograft model. These observations illustrate the versatility of our retargeted HSV platform as it allows expansion of the oncolytic virus toolbox for the treatment of diverse cancers.

Neuroblastomas vary widely in their sensitivities to herpes simplex virotherapy unrelated to virus receptors and susceptibility.

Although most high-risk neuroblastomas are responsive to chemotherapy, relapse is common and long-term survival is < 40%, underscoring the need for more effective treatments. We evaluated the responsiveness of 12 neuroblastoma cell lines to the Δγ134.5 attenuated oncolytic herpes simplex virus (oHSV), Seprehvir (HSV1716), which is currently used in pediatric phase I trials. We found that entry of Seprehvir in neuroblastoma cells is independent of the expression of nectin-1 and the sum of all four known major HSV entry receptors. We observed varying levels of sensitivity and permissivity to Seprehvir, suggesting that the cellular anti-viral response, not virus entry, is the key determinant of efficacy with this virus. In vivo, we found significant anti-tumor efficacy following Seprehvir treatment, which ranged from 6/10 complete responses in the CHP-134 model to a mild prolonged median survival in the SK-N-AS model. Taken together, these data suggest that anti-tumor efficacy cannot be solely predicted based on in vitro response. Whether or not this discordance holds true for other viruses or tumor types is unknown. Our results also suggest that profiling the expression of known viral entry receptors on neuroblastoma cells may not be entirely predictive of their susceptibility to Seprehvir therapy.

Relief of pain induced by varicella-zoster virus in a rat model of post-herpetic neuralgia using a herpes simplex virus vector expressing enkephalin.

Acute and chronic pain (post-herpetic neuralgia or PHN) are encountered in patients with herpes zoster that is caused by reactivation of varicella-zoster virus (VZV) from a state of neuronal latency. PHN is often refractory to current treatments, and additional strategies for pain relief are needed. Here we exploited a rat footpad model of PHN to show that herpes simplex virus (HSV) vector-mediated gene delivery of human preproenkephalin (vHPPE) effectively reduced chronic VZV-induced nocifensive indicators of pain. VZV inoculated at the footpad induced prolonged mechanical allodynia and thermal hyperalgesia that did not develop in controls or with ultraviolet light-inactivated VZV. Subsequent footpad administration of vHPPE relieved VZV-induced pain behaviors in a dose-dependent manner for extended periods, and prophylactic vector administration prevented VZV-induced pain from developing. Short-term pain relief following low-dose vHPPE administration could be effectively prolonged by vector re-administration. HPPE transcripts were increased three- to fivefold in ipsilateral ganglia, but not in the contralateral dorsal root ganglia. VZV hypersensitivity and its relief by vHPPE were not affected by peripheral delivery of opioid receptor agonist or antagonist, suggesting that the efficacy was mediated at the ganglion and/or spinal cord level. These results support further development of ganglionic expression of enkephalin as a novel treatment for the pain associated with Zoster.

Effect of herpes simplex virus vector-mediated interleukin-4 gene therapy on bladder overactivity and nociception.

We investigated the effects of replication-defective herpes simplex virus (HSV) vector expression of interleukin-4 (IL-4) on bladder overactivity and nociception. HSV vector expressing murine interleukin-4 (S4IL4) or the control vector expressing ß-galactosidase (SHZ) were injected to the rat bladder wall. At 1 week after viral injection, in cystometry performed under urethane anesthesia, the S4IL4-treated group did not show the intercontraction intervals reduction during intravesical administration of 10 nM resiniferatoxin (RTx). At 2 weeks after viral injection, behavioral studies were performed on vector-injected animals in an awakened state. Freezing behavior induced by 3 µM RTx, administered for 1 min into the bladder, was significantly suppressed in the S4IL4 group compared with the SHZ group. Murine IL-4 levels examined by ELISA were significantly increased in bladder and bladder afferent dorsal root ganglia at 2 weeks after viral injection. The expression of IL-1ß and IL-2 and bladder inflammatory responses were significantly suppressed in the RTx-irritated bladder of S4IL4-injected rats. These results indicate that HSV vector-mediated interleukin-4 expression in the bladder and bladder afferent pathways reduces the inflammatory response, bladder overactivity and nociceptive behavior induced by bladder irritation in the rat model. Therefore, IL-4 gene therapy could be a new strategy for treating urinary frequency and/or bladder pain.

Expression of HSV-1 receptors in EBV-associated lymphoproliferative disease determines susceptibility to oncolytic HSV.

Epstein-Barr virus (EBV)-associated B-cell lymphoproliferative disease (LPD) after hematopoietic stem cell or solid organ transplantation remains a life-threatening complication. Expression of the virus-encoded gene product, EBER, has been shown to prevent apoptosis via blockade of PKR activation. As PKR is a major cellular defense against Herpes simplex virus (HSV), and oncolytic HSV-1 (oHSV) mutants have shown promising antitumor efficacy in preclinical models, we sought to determine whether EBV-LPD cells are susceptible to infection by oHSVs. We tested three primary EBV-infected lymphocyte cell cultures from neuroblastoma (NB) patients as models of naturally acquired EBV-LPD. NB12 was the most susceptible, NB122R was intermediate and NB88R2 was essentially resistant. Despite EBER expression, PKR was activated by oHSV infection. Susceptibility to oHSV correlated with the expression of the HSV receptor, nectin-1. The resistance of NB88R2 was reversed by exogenous nectin-1 expression, whereas downregulation of nectin-1 on NB12 decreased viral entry. Xenografts derived from the EBV-LPDs exhibited only mild (NB12) or no (NB88R2) response to oHSV injection, compared with a NB cell line that showed a significant response. We conclude that EBV-LPDs are relatively resistant to oHSV virotherapy, in some cases, due to low virus receptor expression but also due to intact antiviral PKR signaling.

Viral vectors for gene therapy: the art of turning infectious agents into vehicles of therapeutics.

Considered by some to be among the simpler forms of life, viruses represent highly evolved natural vectors for the transfer of foreign genetic information into cells. This attribute has led to extensive attempts to engineer recombinant viral vectors for the delivery of therapeutic genes into diseased tissues. While substantial progress has been made, and some clinical successes are over the horizon, further vector refinement and/or development is required before gene therapy will become standard care for any individual disorder.

Engineering herpes simplex virus vectors for gene transfer to neurons.

HSV has a unique relationship with neurons which the virus naturally utilizes for efficient gene transfer and long-term gene expression. Progress in developing defective viral mutants with reduced cytotoxicity, and increasing insight into the state of the viral genome during latency and the functional elements of the latency promoter system, suggest that HSV vectors may be developed in which these natural features are exploited for effective transgene expression in brain.

Ectopic matrix metalloproteinase-9 expression in human brain tumor cells enhances oncolytic HSV vector infection.

Oncolytic herpes simplex virus (oHSV) vectors have shown promise in the treatment of patients with recurrent brain tumors although few complete responses have accrued. Impediments to effective therapy include limited vector distribution on delivery, a consequence of injected virion particle trapping in the tumor extracellular matrix (ECM). To enhance virus delivery and spread, we investigated the use of the matrix metalloproteinase-9 (MMP-9) as a means to degrade collagen type IV, a major component of the ECM and basement membranes of gliomas that is absent in normal brain tissue. SK-N-AS neuroblastoma cells were transduced for constitutive, elevated expression of MMP-9, which did not enhance tumor cell migration in vitro or tumor progression in a murine xenograft brain tumor model. MMP-9 expression improved the distribution and infection of oHSV vectors in spheroid model in vitro. Furthermore, MMP9 induced a vector infection over larger areas of brain tumors in vivo. These results suggest that vector delivery and distribution in vivo can be improved by compromising the ECM, potentially enhancing oncolytic efficacy.

Relationship between expression of herpes simplex virus glycoproteins and susceptibility of target cells to human natural killer activity.

Cells normally insensitive to human natural killer (NK) activity were rendered susceptible by infection with HSV-1. The cytotoxic effector cell was a nonadherent, non-T, non-B lymphocyte. Antibody plus complement treatment, using a monoclonal antibody that recognizes an antigen present on NK cells, removed much of the cytotoxic activity, and a density gradient fraction enriched for NK cells yielded cells of increased virus-specific cytotoxicity. It was concluded that the effector cell active against infected targets possessed characteristics of an NK cell. Blockage of viral protein synthesis during infection inhibited development of increased susceptibility of infected targets to NK activity. When targets were infected with a mutant virus unable to produce viral glycoprotein C (gC), NK activity against these targets was reduced approximately 30% compared with activity against targets infected with wild-type virus. Similarly, activity against targets infected in the presence of 2-deoxyglucose (2dG), which prevents cell surface expression of viral glycoprotein B (gB), was also reduced approximately 30%. An approximately 60% reduction in activity was seen against targets infected with mutant virus in the presence of 2dG; these targets express gD, but neither gB nor gC. When cells expressing various combinations of HSV-1 glycoproteins were used as both labeled targets and cold target competitors, it was found that the susceptibility of a particular target to NK activity was paralleled by its ability to act as a cold target competitor. This indicates that targets with decreased sensitivity to NK cells were less able to bind NK effectors. Further, the amount of interferon produced in co-cultures of NK effectors and infected target cells did not directly correlate with the amount of NK activity generated, and interferon pretreatment of effectors did not decrease virus-specific cytotoxicity. The present results suggest that HSV-1 glycoproteins expressed at the surface of infected targets may act as recognition structures for NK cells.

HSV vector-mediated modification of primary nociceptor afferents: an approach to inhibit chronic pain.

Chronic pain is a serious medical condition with millions of sufferers for whom long-term therapies are either lacking or inadequate. Here we review the use of herpes simplex virus vectors as therapeutic tools to treat chronic pain by gene therapy. We describe an approach to inhibit chronic pain signaling whereby vector-mediated genes transferred to sensory nerves will modify the primary afferent nociceptor to prevent pain signaling to second-order nerves in the spinal cord. This approach may be used to reverse the chronic pain state of the nociceptor and could affect downstream pain-related changes in the central nervous system.

Herpes simplex virus vector-mediated gene delivery for the treatment of lower urinary tract pain.

Interstitial cystitis (IC)/painful bladder syndrome (PBS) is a painful debilitating chronic visceral pain disorder of unknown etiology that affects an estimated 1 million people in the United States alone. It is characterized by inflammation of the bladder that results in chronic pelvic pain associated with bladder symptoms of urinary frequency and urgency. Regardless of the etiology, IC/PBS involves either increased and/or abnormal activity in afferent nociceptive sensory neurons. Pain-related symptoms in patients with IC/PBS are often very difficult to treat. Both medical and surgical therapies have had limited clinical utility in this debilitating disease and numerous drug treatments, such as heparin, dimethylsulfoxide and amitriptyline, have proven to be palliative at best, and in some IC/PBS patients provide no relief whatsoever. Although opiate narcotics have been employed to help alleviate IC/PBS pain, this strategy is fraught with problems as systemic narcotic administration causes multiple unwanted side effects including mental status change and constipation. Moreover, chronic systemic narcotic use leads to dependency and need for dose escalation due to tolerance; therefore, new therapies are desperately needed to treat refractory IC/PBS. This has led our group to develop a gene therapy strategy that could potentially alleviate chronic pelvic pain using the herpes simplex virus-directed delivery of analgesic proteins to the bladder.

Herpes simplex virus vector-mediated delivery of neurturin rescues erectile dysfunction of cavernous nerve injury.

Neurturin (NTN), a member of glial cell line-derived neurotrophic factor (GDNF) family, is known as an important neurotrophic factor for penis-projecting neurons. We recently demonstrated significant protection from erectile dysfunction (ED) following a replication-defective herpes simplex virus (HSV) vector-mediated GDNF delivery to the injured cavernous nerve. Herein, we applied HSV vector-mediated delivery of NTN to this ED model. Rat cavernous nerve was injured bilaterally using a clamp and dry ice. For HSV-treated groups, 20 microl of vector stock was administered directly to the damaged nerve. Delivery of an HSV vector expressing both green fluorescent protein and lacZ (HSV-LacZ) was used as a control. Intracavernous pressure along with systemic arterial pressure (ICP/AP) was measured 2 and 4 weeks after the nerve injury. Fluorogold (FG) was injected into the penile crus 7 days before being killed to assess neuronal survival. Four weeks after nerve injury, rats treated with HSV-NTN exhibited significantly higher ICP/AP compared with untreated or control vector-treated groups. The HSV-NTN group had more FG-positive major pelvic ganglion neurons than the control group following injury. HSV vector-mediated delivery of NTN could be a viable approach for the improvement of ED following cavernous nerve injury.

Herpes simplex virus vector-mediated gene delivery of glutamic acid decarboxylase reduces detrusor overactivity in spinal cord-injured rats.

We examined whether replication-defective herpes simplex virus (HSV) vectors encoding the 67 kDa form of the glutamic acid decarboxylase (GAD(67)) gene product, the gamma-aminobutyric acid (GABA) synthesis enzyme, can suppress detrusor overactivity (DO) in rats with spinal cord injury (SCI). One week after spinalization, HSV vectors expressing GAD and green fluorescent protein (GFP) (HSV-GAD) were injected into the bladder wall. Rats with SCI without HSV injection (HSV-untreated) and those injected with lacZ-encoding reporter gene HSV vectors (HSV-LacZ) were used as controls. Three weeks after viral injection, continuous cystometry was performed under awake conditions in all three groups. In the HSV-GAD group, the number and amplitude of non-voiding contractions (NVCs) were significantly decreased (40-45% and 38-40%, respectively) along with an increase in voiding efficiency, compared with HSV-untreated and HSV-LacZ groups, but micturition pressure was not different among the three groups. Intrathecal application of bicuculline partly reversed the decreased number and amplitude of NVCs, and decreased voiding efficiency in the HSV-GAD group. In the HSV-GAD group, GAD(67) mRNA and protein levels were significantly increased in the L6-S1 dorsal root ganglia (DRG) compared with the HSV-LacZ group, while 57% of DRG cells were GFP-positive, and these neurons showed increased GAD(67)-like immunoreactivity compared with the HSV-LacZ group. These results indicate that GAD gene therapy effectively suppresses DO after SCI predominantly through the activation of spinal GABA(A) receptors. Thus, HSV-based GAD gene transfer to bladder afferent pathways may represent a novel approach for treatment of neurogenic DO.

Rapid identification of nonessential genes of herpes simplex virus type 1 by Tn5 mutagenesis.

The large genome of herpes simplex virus type of (HSV-1) encodes at least 80 polypeptides, the majority of which have no recognized function. A subgroup of these gene products appears to be nonessential for virus replication in cell culture, but contributes to the complex life cycle of the virus in the host. To identify such functions, a simple insertional mutagenesis method has been used for selective inactivation of individual HSV-1 genes. The bacterial transposon Tn5 was allowed to insert randomly into cloned restriction fragments representing the entire short unique (US) region of the HSV-1 genome. Of the 12 open reading frames that were mutagenized with Tn5, mutant derivatives of US2, US4, and US5 were recombined into the virus. These three genes proved to be nonessential for HSV-1 replication in Vero (African Green monkey kidney) cells and the US4 gene appeared to be involved in viral pathogenesis in the central nervous system of mice. This rapid mutagenesis procedure should prove useful in exploring the entire HSV-1 genome as well as the genomes of other complex animal viruses.

A Novel Oncolytic Herpes Simplex Virus Design based on the Common Overexpression of microRNA-21 in Tumors.

BACKGROUND: Recognition sequences for microRNAs (miRs) that are down-regulated in tumor cells have recently been used to render lytic viruses tumor-specific. Since different tumor types down-regulate different miRs, this strategy requires virus customization to the target tumor. We have explored a feature that is shared by many tumor types, the up-regulation of miR-21, as a means to generate an oncolytic herpes simplex virus (HSV) that is applicable to a broad range of cancers. METHODS: We assembled an expression construct for a dominant-negative (dn) form of the essential HSV replication factor UL9 and inserted tandem copies of the miR-21 recognition sequence (T21) in the 3' untranslated region. Bacterial Artificial Chromosome (BAC) recombineering was used to introduce the dnUL9 construct with or without T21 into the HSV genome. Virus was produced by transfection and replication was assessed in different tumor and control cell lines. RESULTS: Virus production was conditional on the presence of the T21 sequence. The dnUL9-T21 virus replicated efficiently in tumor cell lines, less efficiently in cells that contained reduced miR-21 activity, and not at all in the absence of miR-21. CONCLUSION: miR-21-sensitive expression of a dominant-negative inhibitor of HSV replication allows preferential destruction of tumor cells in vitro. This observation provides a basis for further development of a widely applicable oncolytic HSV.

Involvement of a high-mobility-group protein in the transcriptional activity of herpes simplex virus latency-active promoter 2.

Latency-active promoter 2 (LAP 2) is a TATA-less promoter in herpes simplex virus type 1 (HSV-1) that can express genes during viral latency. Four regions of LAP2 are protected from DNase I digestion in vitro by either HeLa cell nuclear extracts or purified Sp1. Transient gene expression assays of LAP2 substitution mutants demonstrate that two of the regions protected by Sp1 and three other regions protected by nuclear extract are important for promoter function. The mutation causing the most significant reduction in expression alters a stretch of 23 thymidine residues (T23) that binds a protein with several properties common to high-mobility-group (HMG) proteins. The T23 binding activity is heat stable, can be inhibited by poly(dA-dT).poly(dA-dT), and is inhibited by minor-groove-binding drugs. Antiserum directed against HMG I(Y) blocked the formation of one of the DNA-protein complexes on the T23 oligonucleotide, suggesting that a protein antigenically related to HMG I(Y) binds to LAP2 in vitro. Direct evidence of HMG I(Y) involvement in LAP2 function is provided by the findings that recombinant HMG I(Y) protein facilitates Sp1 binding to LAP2 in mobility shift assays and that antisense HMG I(Y) RNA specifically inhibits LAP2 function in vivo. These results suggest that DNA structure may be an important determinant of the activity of a promoter that is capable of escaping the global shutoff of transcription that occurs during viral latency.