Copper in serum supports angiogenesis and inhibits replication of wild type HSV-1. Copper chelation is currently being investigated as an anti-angiogenic and anti-neoplastic agent in patients diagnosed with cancer. Herpes simplex virus derived oncolytic viruses (oHSVs) are being evaluated for safety and efficacy in patients, but several host barriers limit their efficacy. Here, we tested if copper inhibits oHSV infection and replication and if copper chelation would augment therapeutic efficacy of oHSV.
Subcutaneous and intracranial tumor bearing mice were treated with oHSV ± ATN-224 to evaluate tumor burden and survival. Virus replication and cell killing was measured in the presence or absence of the copper chelating agent ATN-224 and in the presence or absence of copper in vitro. Micro-vessel density and changes in perfusion were evaluated by immuno-histochemistry and DCE-MRI. Serum stability of oHSV was measured in mice fed with ATN-224. Tumor bearing mice were injected intravenously with oHSV; tumor burden and amount of virus in tumor tissue was evaluated.
Combination of systemic ATN-224 and oHSV significantly reduced tumor growth and prolonged animal survival. Immunohistochemistry and DCE-MRI imaging confirmed that ATN-224 reduced oHSV-induced blood vessel density and vascular leakage. Copper at physiologically relevant concentrations inhibited oHSV replication and glioma cell killing and this effect was rescued by ATN-224. ATN-224 increased serum stability of oHSV and enhanced the efficacy of systemic delivery.
This study shows that combining ATN-224 with oHSV, significantly increased serum stability of oHSV and greatly enhanced its replication and antitumor efficacy.
Glioblastoma (GBM); Oncolytic Virus (OV); Herpes Simplex Virus-1 (HSV-1); ATN-224; Copper ions (Copper)
Both entry and cell-to-cell spread of herpes simplex virus (HSV) involve a cascade of cooperative interactions among the essential glycoproteins D, B, and H/L (gD, gB, and gH/gL, respectively) initiated by the binding of gD to a cognate HSV entry receptor. We previously reported that a variant (D285N/A549T) of glycoprotein B (gB:NT) enabled primary virus entry into cells that were devoid of typical HSV entry receptors. Here, we compared the activities of the gB:NT variant with those of a newly selected variant of glycoprotein H (gH:KV) and a frequently coselected gB variant (gB:S668N). In combination, gH:KV and gB:S668N enabled primary virus entry into cells that lacked established HSV entry receptors as efficiently as did gB:NT, but separately, each variant enabled only limited entry. Remarkably, gH:KV uniquely facilitated secondary virus spread between cells that lacked canonical entry receptors. Transient expression of the four essential entry glycoproteins revealed that gH:KV, but not gB:NT, induced fusion between cells lacking the standard receptors. Because the involvement of gD remained essential for virus spread and cell fusion, we propose that gH:KV mimics a transition state of gH that responds efficiently to weak signals from gD to reach the active state. Computational modeling of the structures of wild-type gH and gH:KV revealed relatively subtle differences that may have accounted for our experimental findings. Our study shows that (i) the dependence of HSV-1 entry and spread on specific gD receptors can be reduced by sequence changes in the downstream effectors gB and gH, and (ii) the relative roles of gB and gH are different in entry and spread.
Sox11 is a high mobility group (HMG) containing transcription factor that is significantly elevated in peripheral neurons in response to nerve injury. In vitro and in vivo studies support a central role for Sox11 in adult neuron growth and survival following injury. Brain-derived neurotrophic factor (BDNF) is a pleiotropic growth factor that has effects on neuronal survival, differentiation, synaptic plasticity and regeneration. BDNF transcription is elevated in the DRG following nerve injury in parallel with Sox11 allowing for the possible regulation by Sox11. To begin to assess the possible influence of Sox11 we used reverse transcriptase PCR assays to determine the relative expression of the nine (I-IXa) noncoding exons and one coding exon (exon IX) of the BDNF gene after sciatic nerve axotomy in the mouse. Exons with upstream promoter regions containing the Sox binding motif 5′-AACAAAG-3′ (I, IV, VII and VIII) were increased at 1d or 3d following axotomy. Exons 1 and IV showed the greatest increase and only exon 1 remained elevated at 3d. Luciferase assays showed that Sox11 could activate the most highly regulated exons, I and IV, and that this activation was reduced by mutation of putative Sox binding sites. Exon expression in injured DRG neurons had some overlap with Neuro2a cells that overexpress Sox11, showing elevation in exon IV and VII transcripts. These findings indicate cell type and contextual specificity of Sox11 in modulation of BDNF transcription.
sensory neuron; nerve regeneration; BDNF; neurotrophin; sry
Oncolytic viral therapy has been explored widely as an option for glioma treatment but its effectiveness has remained limited. CCN1 is an extracellular matrix (ECM) protein elevated in cancer cells that modulates their adhesion and migration by binding cell surface receptors. In this study, we examined an hypothesized role for CCN1 in limiting the efficacy of oncolytic viral therapy for glioma, based on evidence of CCN1 induction that occurs in this setting. Strikingly, we found that exogenous CCN1 in glioma ECM orchestrated a cellular antiviral response that reduced viral replication and limited cytolytic efficacy. Gene expression profiling and real time PCR analysis revealed a significant induction of type-I interferon responsive genes in response to CCN1 exposure. This induction was accompanied by activation of the Jak/Stat signaling pathway, consistent with induction of an innate antiviral cellular response. Both effects were mediated by the binding of CCN1 to the cell surface integrin α6β1, activating its signaling and leading to rapid secretion of interferon-α, which was essential for the innate antiviral effect. Together, our findings reveal how an integrin signaling pathway mediates activation of a type-I antiviral interferon response that can limit the efficacy of oncolytic viral therapy. Further, they suggest therapeutic interventions to inhibit CCN1-integrin α6 interactions to sensitize gliomas to viral oncolysis.
CCN1; Cyr61; Glioma; Oncolytic Virus
Factors that enhance the intrinsic growth potential of adult neurons are key players in the successful repair and regeneration of neurons following injury. Injury-induced activation of transcription factors has a central role in this process because they regulate expression of regeneration-associated genes. Sox11 is a developmentally expressed transcription factor that is significantly induced in adult neurons in response to injury. Its function in injured neurons is however undefined. Here, we report studies that use herpes simplex virus (HSV)-vector-mediated expression of Sox11 in adult sensory neurons to assess the effect of Sox11 overexpression on neuron regeneration. Cultured mouse dorsal root ganglia (DRG) neurons transfected with HSV-Sox11 exhibited increased neurite elongation and branching relative to naïve and HSV-vector control treated neurons. Neurons from mice injected in foot skin with HSV-Sox11 exhibited accelerated regeneration of crushed saphenous nerves as indicated by faster regrowth of axons and nerve fibers to the skin, increased myelin thickness and faster return of nerve and skin sensitivity. Downstream targets of HSV-Sox11 were examined by analyzing changes in gene expression of known regeneration-associated genes. This analysis in combination with mutational and chromatin immunoprecipitation assays indicates that the ability of Sox11 to accelerate in vivo nerve regeneration is dependent on its transcriptional activation of the regeneration-associated gene, small proline rich protein 1a (Sprr1a). This finding reveals a new functional linkage between Sox11 and Sprr1a in adult peripheral neuron regeneration.
transcriptional control; sry; skin delivery; saphenous nerve injury; HSV vector
Successful oncolytic virus treatment of malignant glioblastoma multiforme depends on widespread tumor-specific lytic virus replication and escape from mitigating innate immune responses to infection. Here we characterize a new HSV vector, JD0G, that is deleted for ICP0 and the joint sequences separating the unique long and short elements of the viral genome. We observed that JD0G replication was enhanced in certain glioblastoma cell lines compared to HEL cells, suggesting that a vector backbone deleted for ICP0 may be useful for treatment of glioblastoma. The innate immune response to virus infection can potentially impede oncolytic vector replication in human tumors. Indoleamine-2,3-dioxygenase (IDO) is expressed in response to interferon γ (IFNγ) and has been linked to both antiviral functions and to the immune escape of tumor cells. We observed that IFNγ treatment of human glioblastoma cells induced the expression of IDO and that this expression was quelled by infection with both wild-type and JD0G viruses. The role of IDO in inhibiting virus replication and the connection of this protein to the escape of tumor cells from immune surveillance suggest that IDO downregulation by HSV infection may enhance the oncolytic activity of vectors such as JD0G.
Preclinical evidence indicates that gene transfer to the dorsal root ganglion (DRG) using replication defective herpes simplex virus (HSV)-based vectors can reduce pain-related behavior in animal models of pain. This clinical trial was carried out to assess the safety and explore the potential efficacy of this approach in humans.
We conducted a multicenter, dose-escalation, Phase I clinical trial of NP2, a replication defective HSV-based vector expressing human preproenkephalin (PENK) in subjects with intractable focal pain caused by cancer. NP2 was injected intradermally into the dermatome(s) corresponding to the radicular distribution of pain. The primary outcome was safety. As secondary measures, efficacy of pain relief was assessed using a numeric rating scale (NRS), the Short Form McGill Pain Questionnaire (SF-MPQ) and concurrent opiate usage.
Ten subjects with moderate to severe intractable pain despite treatment with more than 200 mg/day of morphine (or equivalent) were enrolled into the study. Treatment was well tolerated with no study agent-related serious adverse events (SAE) observed at any point in the study. Subjects receiving the low dose of NP2 reported no substantive change in pain. Subjects in the middle and high dose cohorts reported pain relief as assessed by NRS and SF-MPQ.
Treatment of intractable pain with NP2 was well tolerated. There were no placebo controls in this relatively small study, but the dose-responsive analgesic effects suggest that NP2 may be effective in reducing pain and warrants further clinical investigation.
Peripheral neuropathy is a common aging-related degenerative disorder that interferes with daily activities and leads to increased risk of falls and injury in the elderly. The etiology of most aging-related peripheral neuropathy is unknown. Inherited defects in several genome maintenance mechanisms cause tissue-specific accelerated aging, including neurodegeneration. We tested the hypothesis that a murine model of XFE progeroid syndrome, caused by reduced expression of ERCC1-XPF DNA repair endonuclease, develops peripheral neuropathy. Nerve conduction studies revealed normal nerve function in young adult (8 week) Ercc1−/Δ mice, but significant abnormalities in 20 week-old animals. Morphologic and ultrastructural analysis of the sciatic nerve from mutant mice revealed significant alterations at 20 but not 8 weeks of age. We conclude that Ercc1−/Δ mice have accelerated spontaneous peripheral neurodegeneration that mimics aging-related disease. This provides strong evidence that DNA damage can drive peripheral neuropathy and offers a rapid and novel model to test therapies.
Xeroderma pigmentosum; progeria; neurodegeneration; DNA repair; nerve conduction; nerve morphology
Hutchinson-Gilford progeria syndrome (HGPS) is a genetic disease in which children develop pathologies associated with old age. HGPS is caused by a mutation in the LMNA gene, resulting in the formation of a dominant negative form of the intermediate filament, nuclear structural protein lamin A, termed progerin. Expression of progerin alters the nuclear architecture and heterochromatin, affecting cell cycle progression and genomic stability. Two groups recently reported the successful generation and characterization of induced pluripotent stem cells (iPSCs) from HGPS fibroblasts. Remarkably, progerin expression and senescence phenotypes are lost in iPSCs but not in differentiated progeny. These new HGPS iPSCs are valuable for characterizing the role of progerin in driving HGPS and aging and for screening therapeutic strategies to prevent or delay cell senescence.
Herpes simplex virus type 1 (HSV-1) entry into permissive cells involves attachment to cell-surface glycosaminoglycans (GAGs) and fusion of the virus envelope with the cell membrane triggered by the binding of glycoprotein D (gD) to cognate receptors. In this study, we characterized the observation that soluble forms of the gD ectodomain (sgD) can mediate entry of gD-deficient HSV-1. We examined the efficiency and receptor specificity of this activity and used sequential incubation protocols to determine the order and stability of the initial interactions required for entry. Surprisingly, virus binding to GAGs did not increase the efficiency of sgD-mediated entry and gD-deficient virus was capable of attaching to GAG-deficient cells in the absence of sgD. These observations suggested a novel binding interaction that may play a role in normal HSV infection.
HSV-1; glycoprotein D (gD); glycosaminoglycans; nectin-1; HVEM; soluble glycoprotein; virus entry
Herpes simplex virus entry into cells requires the binding of envelope glycoprotein D (gD) to an entry receptor. Depending on the cell, entry occurs by different mechanisms, including fusion at the cell surface or endocytosis. Here we examined the entry mechanism through a non-HSV receptor mediated by a soluble bi-specific adapter protein composed of recognition elements for gD and the EGF receptor (EGFR). Virus entered into endosomes using either EGF or an EGFR-specific single chain antibody (scFv) for receptor recognition. Infection was less efficient with the EGF adapter which could be attributed to its weaker binding to viral gD. Infection mediated by the scFv adapter was pH sensitive, indicating that gD-EGFR bridging alone was insufficient for capsid release from endosomes. We also show that the scFv adapter enhanced infection of EGFR-expressing tumor tissue in vivo. Our results indicate that adapters may retarget HSV infection without drastically changing the entry mechanism.
HSV entry; bispecific adapter; EGF receptor; endocytosis; fusion
Oncolytic HSV (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 (MMP9) 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 MMP9, which did not enhance tumor cell migration in vitro or tumor progression in a murine xenograft brain tumor model. MMP9 expression afforded increased distribution of oHSV vector-infected tumor cell spheroids and afforded 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.
We investigated whether replication-defective herpes simplex virus (HSV) vectors encoding genes of glutamic acid decarboxylase (GAD), the gamma-aminobutyric acid synthesis enzyme, can suppress detrusor-sphincter dyssynergia (DSD) in rats with spinal cord injury (SCI).
Materials and Methods
One week after spinalization, HSV vectors expressing GAD and green fluorescent protein (HSV-GAD) were injected to the bladder wall. SCI rats without HSV injection (sham) and those injected with LacZ-encoding HSV vectors (HSV-LacZ) were used as controls. Three weeks after viral injection, simultaneous recordings of urethral pressure and intravesical pressure were performed under an awake condition in three groups.
In the HSV-GAD group, the urethral pressure rise during bladder contractions was significantly reduced by 77–79% compared with sham or HSV-LacZ groups, but bladder activity and urethral baseline pressure were not different among three groups. Intrathecal application of bicuculline, a GABAA antagonist, almost completely reversed the decrease in urethral pressure rise during bladder contractions whereas intrathecal saclofen, a GABAB antagonist, partially reversed it. In the HSV-GAD group, GAD67 mRNA was significantly increased in L6-S1 dorsal root ganglia, where bladder afferents originate, compared with the HSV-LacZ group.
HSV-based GAD gene transfer to bladder afferent pathway may represent a novel approach for the treatment of DSD in SCI.
gene therapy; detrusor overactivity; detrusor-sphincter dyssynergia; GABA; spinalized; C-fiber bladder afferents
Importance of the field
Erectile dysfunction (ED) is a major men’s health problem. Although the high success rate of treating ED by phosphodiesterase 5 (PDE5) inhibitors has been reported, there are a significant number of ED patients who do not respond to currently available treatment modalities.
Areas covered in this review
To understand the current status of gene therapy application for ED, gene therapy approaches for ED treatment are reviewed.
What the reader will gain
Gene therapy strategies that can enhance nitric oxide (NO) production or NO-mediated signaling pathways, growth factor-mediated nerve regeneration or K+ channel activity in the smooth muscle could be promising approaches for the treatment of ED. Although the majority of gene therapy studies are still in the preclinical phase, the first clinical trial using non-viral gene transfer of Ca2+-activated, large-conductance K+ channels into the corpus cavernosum of ED patients showed positive results.
Take home message
Gene therapy represents an exciting future treatment option for ED, especially for people with severe ED unresponsive to current first-line therapies such as PDE5 inhibitors although the long-term safety of both viral and non-viral gene therapies should be established.
gene therapy; erectile dysfunction; nitric oxide; growth factor; K+ channel
Pathological alterations of ion channel activity result from changes in modulatory mechanisms governing receptor biology. Here we describe a conditional herpes simplex virus (HSV) replication–based strategy to discover channel modulators whereby inhibition of agonist-induced channel activation by a vector-expressed modulatory gene product prevents ion flux, osmotic shock and cell death. Inhibition of channel activity, in this case, the rat vanilloid (Trpv1) or the glycine receptor (GlyRα1), can allow selection of escape vector plaques containing the ‘captured’ modulatory gene for subsequent identification and functional analysis. We validated this prediction using mixed infections of a wild-type Trpv1 expression vector vTTHR and a nonfunctional ‘poreless’ Trpv1 subunit–expressing vector, vHP, wherein vHP was highly selected from a large background of vTTHR viruses in the presence of the Trpv1 agonist, capsaicin. The approach should be useful for probing large libraries of vector-expressed cDNAs for the presence of ion channel modulators.
Phosphorylation of the vanilloid receptor (TRPV1) by protein kinase C epsilon (PKCε) plays an important role in the development of chronic pain. Here, we employ a highly defective herpes simplex virus vector (vHDNP) that expresses dominant negative PKCε (DNPKCε) as a strategy to demonstrate that PKCε is essential for: (i) maintenance of basal phosphorylation and normal TRPV1 responses to capsaicin (CAPS), a TRPV1 agonist and (ii) enhancement of TRPV1 responses by phorbol esters. Phorbol esters induced translocation of endogenous PKCε to the plasma membrane and thereby enhanced CAPS currents. These results were extended to an in-vivo pain model in which vHDNP delivery to dorsal root ganglion neurons caused analgesia in CAPS-treated, acutely inflamed rat hind paws. These findings support the conclusion that in addition to receptor sensitization, PKCε is essential for normal TRPV1 responses in vitro and in vivo.
capsaicin; desensitization; HSV; modulation; nociception; PKC; TRPV1
Herpes simplex virus (HSV) entry into cells is triggered by the binding of envelope glycoprotein D (gD) to a specific receptor, such as nectin-1 or herpesvirus entry mediator (HVEM), resulting in activation of the fusion effectors gB and gH and virus penetration. Here we report the identification of a hyperactive gB allele, D285N/A549T, selected by repeat passage of a gD mutant virus defective for nectin-1 binding through cells that express a gD-binding-impaired mutant nectin-1. The gB allele in a wild-type virus background enabled the use of other nectins as virus entry receptors. In addition, combination of the mutant allele with an epidermal growth factor receptor (EGFR)-retargeted gD gene yielded dramatically increased EGFR-specific virus entry compared to retargeted virus carrying wild-type gB. Entry of the gB mutant virus into nectin-1-bearing cells was markedly accelerated compared to that of wild-type virus, suggesting that the gB mutations affect a rate-limiting step in entry. Our observations indicate that ineffective gD activation can be complemented by hypersensitization of a downstream component of the entry cascade to gD signaling.
To initiate infection, equine herpesvirus type 1 (EHV-1) attaches to heparan sulfate on cell surfaces and then interacts with a putative glycoprotein D receptor(s). After attachment, virus entry occurs either by direct fusion of the virus envelope with the plasma membrane or via endocytosis followed by fusion between the virus envelope and an endosomal membrane. Upon fusion, de-enveloped virus particles are deposited into the cytoplasm and travel to the nucleus for viral replication. In this report, we examined the mechanism of EHV-1 intracellular trafficking and investigated the ability of EHV-1 to utilize specific cellular components to efficiently travel to the nucleus post-entry. Using a panel of microtubule depolymerizing drugs and inhibitors of microtubule motor proteins, we show that EHV-1 infection is dependent on both the integrity of the microtubule network and the minus-end microtubule motor protein, dynein. In addition, we show that EHV-1 actively induces the acetylation of tubulin, a marker of microtubule stabilization, as early as 15 minutes post-infection. Finally, our data support a role for the cellular kinase, ROCK1, in virus trafficking to the nucleus.
EHV-1; trafficking; microtubules; dynein; ROCK1
Viral vector-based gene expression libraries from normal or diseased tissues offer opportunities to interrogate cellular functions that influence or participate directly in specific biological processes. Here we report the creation and characterization of a herpes simplex virus (HSV)-based expression library consisting of cDNAs derived from PC12 pheochromocytoma cells. A replication-defective HSV vector backbone was engineered to contain both a bacterial artificial chromosome (BAC) and the Invitrogen in vitro Gateway recombination system, creating DBAC-GW. A cDNA library was produced and transferred into the DBAC-GW genome by in vitro recombination and selection in bacteria to produce DBAC-L. DBAC-L contained at least 15,000 unique cDNAs, as shown by DNA array analysis of PCR-amplified cDNA inserts, representing a wide range of cancer- and neuron-related cellular functions. Transfection of the recombinant DBAC-L DNA into complementing animal cells produced more than 1 million DBAC-L virus particles representing the library genes. By microarray analysis of vector-infected cells, we observed that individual members of this vector population expressed unique PC12 cDNA-derived mRNA, demonstrating the power of this system to transfer and express a variety of gene activities. We discuss the potential utility of this and similarly derived expression libraries for genome-wide approaches to identify cellular functions that participate in complex host-pathogen interactions or processes related to disease and to cell growth and development.
We examined whether replication-defective herpes simplex virus (HSV) vectors encoding the 67 Kd form of the glutamic acid decarboxylase (GAD67) gene product, the gamma-aminobutyric acid (GABA) synthesis enzyme, can suppress detrusor overactivity (DO) in spinal cord injury (SCI) rats. One week after spinalization, HSV vectors expressing GAD and green fluorescent protein (GFP) (HSV-GAD) were injected into the bladder wall. SCI rats 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, GAD67 mRNA and protein levels were significantly increased in 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 GAD67-like immunoreactivity compared with the HSV-LacZ group. These results indicate that GAD gene therapy effectively suppresses DO following SCI predominantly via activation of spinal GABAA receptors. Thus, HSV-based GAD gene transfer to bladder afferent pathways may represent a novel approach for the treatment of neurogenic DO.
gene therapy; detrusor overactivity; herpes simplex virus; GABA; spinalized; C-fiber bladder afferents
Neurturin (NTN), a member of glial cell line-derived neurotrophic factor (GDNF) family, is known as an important neurotrphic 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, 20μl of vector stock was administered directly to the damaged nerve. Delivery of an HSV vector expressing both green fluorescent protein (GFP) 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 sacrifice to assess neuronal survival. Four weeks after nerve injury, rats treated with HSV-NTN exhibited significantly higher ICP/AP compared to untreated or control vector treated groups. The HSV-NTN group had more FG-positive MPG neurons than control group following injury. HSV vector-mediated delivery of NTN could be a viable approach for improvement of erectile dysfunction following cavernous nerve injury.
Erectile Dysfunction; Neurturin; Gene Therapy; HSV
Interstitial cystitis/painful bladder syndrome (IC/PBS) is a major challenge to treat. We studied the effect of targeted and localized expression of enkephalin in afferent nerves that innervate the bladder by gene transfer using replication-defective herpes simplex virus (HSV) vectors in a rat model of bladder hyperactivity and pain. Replication-deficient HSV vectors encoding preproenkephalin, which is a precursor for Met- and Leu-enkephalin, or control vector encoding the lacZ reporter gene, were injected into the bladder wall of female rats. After viral vector injection, quantitative polymerase chain reaction showed high preproenkephalin transgene levels in bladder and dorsal root ganglia innervating the bladder in enkephalin vector-treated animals. Functionally, enkephalin vector-treated animals showed reductions in bladder hyperactivity and nociceptive behavior induced by intravesical application of capsaicin; however, vector-mediated expression of enkephalin did not alter normal voiding. This antinociceptive effect of enkephalin gene therapy was antagonized by naloxone hydrochloride administration. Together, our results with HSV vectors encoding preproenkephalin demonstrated physiological improvement in visceral pain induced by bladder irritation. Thus, gene therapy may represent a potentially useful treatment modality for bladder hypersensitive disorders such as IC/PBS.
Both initial infection and cell-to-cell spread by herpes simplex virus type 1 (HSV-1) require the interaction of the viral glycoprotein D (gD) with an entry receptor on the cell surface. The two major HSV entry receptors, herpesvirus entry mediator (HVEM) and nectin-1, mediate infection independently but are coexpressed on a variety of cells. To determine if both receptors are active in these instances, we have established mutant viruses that are selectively impaired for recognition of one or the other receptor. In plaque assays, these viruses showed approximately 1,000-fold selectivity for the matched receptor over the mismatched receptor. Separate assays showed that each virus is impaired for both infection and spread through the mismatched receptor. We tested several human tumor cell lines for susceptibility to these viruses and observed that HT29 colon carcinoma cells are susceptible to infection by nectin-1-restricted virus but are highly resistant to HVEM-restricted virus infection, despite readily detectable HVEM expression on the cell surface. HVEM cDNA isolated from HT29 cells rendered HSV-resistant cells permissive for infection by the HVEM-restricted virus, suggesting that HT29 cells lack a cofactor for HVEM-mediated infection or express an HVEM-specific inhibitory factor. Passaging of HVEM-restricted virus on nectin-1-expressing cells yielded a set of gD missense mutations that each restored functional recognition of nectin-1. These mutations identify residues that likely play a role in shaping the nectin-1 binding site of gD. Our findings illustrate the utility of these receptor-restricted viruses in studying the early events in HSV infection.
We examined the analgesic properties of endomorphin-2 expressed in DRG neurons transduced with a non-replicating herpes simplex virus (HSV)-based vector containing a synthetic endomorphin-2 gene construct. HSV-mediated endomorphin-2 expression reduced nocisponsive behaviors in response to mechanical and thermal stimuli after injection of complete Freund’s adjuvant (CFA) into the paw, and reduced peripheral inflammation measured by paw swelling after injection of CFA. The analgesic effect of the vector was blocked by either intraperitoneal or intrathecal administration of naloxone methiodide, blocking peripheral and central μ opioid receptors, respectively. Endomorphin-2 vector injection also reduced spontaneous pain-related behaviors in the delayed phase of the formalin test and in both CFA and formalin models suppressed spinal c-fos expression. The magnitude of the vector-mediated analgesic effect on the delayed phase of the formalin test was similar in naïve animals and in animals with opiate tolerance induced by twice daily treatment with morphine, suggesting that there was no cross-tolerance between vector-mediated endomorphin-2 and morphine. These results suggest that transgene-mediated expression of endomorphin-2 in transduced DRG neurons in vivo acts both peripherally and centrally through mu opioid receptors to reduce pain perception.
Inflammatory pain; Opiates; Endomorphin-2; Gene therapy; Herpes simplex virus
ICP0 is a multi-functional herpes simplex virus type 1 (HSV-1) immediate-early (IE) gene product that contributes to efficient virus growth and reactivation from latency. Here we show that HSV-1-induced cell-cycle arrest at the G2/M border requires ICP0 and Chk2 kinase and that ICP0 expression by transfection or infection induces ATM-dependent phosphorylation of Chk2 and Cdc25C. Infection of cells with a replication-defective mutant virus deleted for all the regulatory IE genes except ICP0 (TOZ22R) induced G2/M arrest whereas a mutant virus deleted in addition for ICP0 (QOZ22R) failed to do so. Chk2-deficient cells and cells expressing a kinase-deficient Chk2 did not undergo cell-cycle arrest in response to TOZ22R infection. Chk2 deficiency diminished the growth of wild-type HSV-1, but not the growth of an ICP0-deleted recombinant virus. Together, these results are consistent with the interpretation that ICP0 activates a DNA damage response pathway to arrest cells in G2/M phase and promote virus growth.
HSV-1; cell cycle; Chk2; DNA damage response; ICP0