HSV Vectors Research Articles

HSV-1 as a Model for Emerging Gene Delivery Vehicles

The majority of viral vectors currently used possess modest cargo capability (up to 40 kb) being based on retroviruses, lentiviruses, adenoviruses, and adenoassociated viruses. These vectors have made the most rapid transition from laboratory to clinic because their small genomes have simplified their characterization and modification. However, there is now an increasing need both in research and therapy to complement this repertoire with larger capacity vectors able to deliver multiple transgenes or to encode complex regulatory regions, constructs which can easily span more than 100 kb. Herpes Simplex Virus Type I (HSV-1) is a well-characterized human virus which is able to package about 150 kb of DNA, and several vector systems are currently in development for gene transfer applications, particularly in neurons where other systems have low efficiency. However, to reach the same level of versatility and ease of use as that of smaller genome viral vectors, simple systems for high-titer production must be developed. This paper reviews the major HSV-1 vector systems and analyses the common elements which may be most important to manipulate to achieve this goal.

A Herpes Simplex Virus-Derived Replicative Vector Expressing LIF Limits Experimental Demyelinating Disease and Modulates Autoimmunity

Herpes simplex virus type 1 (HSV-1) has properties that can be exploited for the development of gene therapy vectors. The neurotropism of HSV enables delivery of therapeutic genes to the nervous system. Using a bacterial artificial chromosome (BAC), we constructed an HSV-1(17+)-based replicative vector deleted of the neurovirulence gene γ134.5, and expressing leukemia inhibitory factor (LIF) as a transgene for treatment of experimental autoimmune encephalomyelitis (EAE). EAE is an inducible T-cell mediated autoimmune disease of the central nervous system (CNS) and is used as an animal model for multiple sclerosis. Demyelination and inflammation are hallmarks of both diseases. LIF is a cytokine that has the potential to limit demyelination and oligodendrocyte loss in CNS autoimmune diseases and to affect the T-cell mediated autoimmune response. In this study SJL/J mice, induced for EAE, were treated with a HSV-LIF vector intracranially and the subsequent changes in disease parameters and immune responses during the acute disease were investigated. Replicating HSV-LIF and its DNA were detected in the CNS during the acute infection, and the vector spread to the spinal cord but was non-virulent. The HSV-LIF significantly ameliorated the EAE and contributed to a higher number of oligodendrocytes in the brains when compared to untreated mice. The HSV-LIF therapy also induced favorable changes in the expression of immunoregulatory cytokines and T-cell population markers in the CNS during the acute disease. These data suggest that BAC-derived HSV vectors are suitable for gene therapy of CNS disease and can be used to test the therapeutic potential of immunomodulatory factors for treatment of EAE.

Noradrenaline increases pain facilitation from the brain during inflammatory pain

Antidepressants that inhibit the recapture of noradrenaline have variable effects in chronic pain which may be related to the complex role of noradrenaline in pain modulation. Whereas at the spinal cord noradrenaline blocks nociceptive transmission, both antinociception and pronociception were reported after noradrenaline release in the brain. To study the role of noradrenaline in pain modulatory areas of the brain, we elected the dorsal reticular nucleus (DRt), a key pain facilitatory area located at the medulla oblongata. Three studies were performed. First, we show that the infusion in the DRt of nomifensine, which increases local extracellular levels of noradrenaline as shown by in vivo microdialysis, also enhances pain behavioral responses during both phases of the formalin test, a classic inflammatory pain model. Then, we demonstrate that the formalin test triggers the release of noradrenaline in the DRt in a biphasic pattern that matches the two phases of the test. Finally, we show that reducing noradrenaline release into the DRt, using an HSV-1 vector which decreases the expression of tyrosine hydroxylase in noradrenergic DRt-projecting neurons, attenuates pain behavioral responses in both phases of the formalin test. The increased noradrenaline levels induced by the infusion of nomifensine at the DRt, along with the hyperalgesic effects of noradrenaline released at the DRt upon noxious stimulation, indicates that noradrenaline may enhance pain facilitation from the brain. It is important to evaluate if antidepressants that inhibit noradrenaline recapture enhance pain facilitation from the brain herein attenuating their analgesic effects.

Performance Efficiency of Quantization using HSV Colour Space and Vector Cosine Angle Distance in CBIR with Different Image Sizes

Content Based Image Retrieval (CBIR) is an active research field in the past decades. Against the traditional system where the images are retrieved based on the key word search, CBIR systems retrieve the images based on the visual content. Even though some of the modern systems like relevance feedback system which improves the performance of CBIR systems exists, the importance of retrieving the images based on the low level features like Colour, Texture and Shape still determine the development of CBIR systems and cannot be undermined. Colour Histograms, Histogram Distance Measurements, Colour Spaces and Quantization play an important role in retrieving images based on similarities. In this paper, a novel method is presented for determining the efficiency of different quantization methods using HSV Colour space and measuring the Vector Cosine Angle distance of the images with different sizes of images like 256 X 256, 128 X 128, 64 X 64, 32 X 32, 16 X 16 and 8 X 8 pixels for efficient image retrieval and comparing the time utilized for retrieval in each sizes and measuring the Overall efficiency.

Herpes Simplex Virus Vector Mediated Gene Therapy of Tumor Necrosis Factor-α Blockade for Bladder Overactivity and Nociception in Rats

We examined the effects of tumor necrosis factor-α blockade on bladder overactivity and nociception using replication defective HSV vectors expressing tumor necrosis factor-α soluble receptor. HSV vectors expressing tumor necrosis factor-α soluble receptor or β-galactosidase/green fluorescent protein as the control were injected into the bladder wall of female Sprague-Dawley® rats. Green fluorescent protein was observed with fluorescent microscopy in the bladder and L6 dorsal root ganglia. mRNA and protein expression of tumor necrosis factor-α, and interleukin-1β and 6 as well as myeloperoxidase activity in the bladder were determined by quantitative reverse transcriptase-polymerase chain reaction and enzyme-linked immunosorbent assay 4 hours after intravesical resiniferatoxin administration. c-Fos positive neurons were counted in the L6 spinal dorsal horn. Cystometry and behavioral analyses were also performed. Green fluorescent protein expression was confirmed in the bladder and L6 dorsal root ganglia. Resiniferatoxin administration significantly increased tumor necrosis factor-α mRNA and protein levels in the bladder in controls. Tumor necrosis factor-α mRNA was also increased in the tumor necrosis factor-α soluble receptor group, although tumor necrosis factor-α protein up-regulation was suppressed. The up-regulation of interleukin-1β and 6 mRNA and protein levels, and the myeloperoxidase activity seen in controls were suppressed in the tumor necrosis factor-α soluble receptor group. c-Fos positive cells in the L6 spinal dorsal horn were less prominent in the tumor necrosis factor-α soluble receptor group than in controls. On cystometry the significant decrease in intercontraction intervals after resiniferatoxin infusion detected in controls was not seen in the tumor necrosis factor-α soluble receptor group. On behavioral analyses freezing behavior was significantly decreased in the tumor necrosis factor-α soluble receptor group without affecting licking behavior. HSV vector mediated tumor necrosis factor-α blockade gene therapy in the bladder and bladder afferent pathways decreases the bladder pain and overactivity induced by nociceptive bladder stimuli.

Early pro-inflammatory host response to recombinant HSV-SIV vaccination in sooty mangabeys

Background Naturally infected sooty mangabeys (SM) do not progress to AIDS unlike SIV-infected non-natural hosts including rhesus macaques (RM). Differences in the innate immune response to SIV may be critical in avoidance of disease progression in SIV-infected SM. In this study we investigated innate and adaptive immune responses to recombinant HSV vectors expressing SIV Gag, Env, and a Rev-Tat-Nef fusion protein in six SIVnegative SM and four RM following single intramuscular inoculation. Methods Plasma samples were analyzed using Luminex 28-plex assay. Innate immune cells and SIV-specific cellular immune responses were investigated by flow cytometry and IFN-g ELISPOT assay. Results A significant transient decline of circulating Natural Killer T (NKT) cells, pDCs, and NK cells was observed in SM on day one post vaccination, with a return to baseline levels by day 3-7. Also an increased activation of SM NK cells was observed that returned to baseline by day 9. In contrast, RM displayed no significant change in circulating NKT cell and NK cell frequencies. Both RM and SM exhibited similar transient increases in plasma levels of several inflammatory cytokines/chemokines including IL-1RA, IFN-g ,I L-6, IL-8, macrophage migration inhibitory factor (MIF), monocyte chemotactic protein 1 (MCP-1), vascular endothelial growth factor (VEGF), and interferon-inducible T cell alpha-chemoattractant (I-TAC) by day one, suggesting activation of multiple immune cells including macrophages and DCs. By day 14, plasma levels of analytes such as IL-6 and Eotaxin persisted at elevated levels in RM, but not SM, suggesting delayed resolution of immune activation in RM. SIV-specific cellular immune responses were detected in both species. Conclusion

Inhibition of Indoleamine-2,3-dioxygenase (IDO) in Glioblastoma Cells by Oncolytic Herpes Simplex Virus

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.

Overexpression of either lysine-specific demethylase-1 or CLOCK, but not Co-Rest, improves long-term expression from a modified neurofilament promoter, in a helper virus-free HSV-1 vector system

Long-term expression from helper virus-free Herpes Simplex Virus (HSV-1) vectors is required for many specific neural gene therapies and studies on neuronal physiology. We previously developed a promoter that supports long-term, neuron-specific expression by fusing the chicken ß-globin insulator (INS), followed by an upstream enhancer from the rat tyrosine hydroxylase (TH) promoter, to a neurofilament heavy gene (NFH) promoter. Here, we examined the capability of specific transcription factors to further improve long-term expression from this promoter. Following a HSV-1 virus infection, the virus genome is localized to promyelocytic leukemia protein (PML) nuclear bodies (NB). At these sites, specific cellular transcription factors interact with HSV-1 encoded transcription factors, and together regulate HSV-1 gene expression. Importantly, lysine-specific demethylase-1 (LSD1), CLOCK, and Co-Rest each activate HSV-1 gene expression. However, gene expression from HSV-1 vectors differs in a number of important aspects from the virus, including no HSV-1 genes are expressed. Nonetheless, these observations raise the possibility that specific transcription factors may improve long-term expression from specific promoters in HSV-1 vectors. Here, we show that overexpression of either LSD1 or CLOCK improves long-term expression from the INS-TH-NFH promoter, but overexpression of Co-Rest supports levels of long-term expression similar to those supported by a control vector. Further, overexpression of LSD1 is compatible with neuron-specific expression. Thus, overexpressing specific transcription factors can improve long-term expression from specific cellular promoters in HSV-1 vectors, and the chromatin structure of the vector has an important role in enabling expression.

Antibody-mediated targeted gene transfer of helper virus-free HSV-1 vectors to rat neocortical neurons that contain either NMDA receptor 2B or 2A subunits

Because of the numerous types of neurons in the brain, and particularly the forebrain, neuron type-specific expression will benefit many potential applications of direct gene transfer. The two most promising approaches for achieving neuron type-specific expression are targeted gene transfer to a specific type of neuron and using a neuron type-specific promoter. We previously developed antibody-mediated targeted gene transfer with Herpes Simplex Virus (HSV-1) vectors by modifying glycoprotein C (gC) to replace the heparin binding domain, which mediates the initial binding of HSV-1 particles to many cell types, with the Staphylococcus A protein ZZ domain, which binds immunoglobulin (Ig) G. We showed that a chimeric gC--ZZ protein is incorporated into vector particles and binds IgG. As a proof-of-principle for antibody-mediated targeted gene transfer, we isolated complexes of these vector particles and an anti-NMDA NR1 subunit antibody, and demonstrated targeted gene transfer to neocortical cells that contain NR1 subunits. However, because most forebrain neurons contain NR1, we obtained only a modest increase in the specificity of gene transfer, and this targeting specificity is of limited utility for physiological experiments. Here, we report efficient antibody-mediated targeted gene transfer to NMDA NR2B- or NR2A-containing cells in rat postrhinal cortex, and a neuron-specific promoter further restricted recombinant expression to neurons. Of note, because NR2A-containing neurons are relatively rare, these results show that antibody-mediated targeted gene transfer with HSV-1 vectors containing neuron type-specific promoters can restrict recombinant expression to specific types of forebrain neurons of physiological significance.

A 16 bp upstream sequence from the rat tyrosine hydroxylase promoter supports long-term expression from a neurofilament promoter, in a helper virus-free HSV-1 vector system

Helper virus-free Herpes Simplex Virus vector-mediated gene transfer has supported studies on neuronal physiology, and may support specific gene therapies. Long-term, neuron-specific expression is required for many of these applications. A neurofilament heavy gene (NFH) promoter does not support long-term expression. We previously developed a promoter that supports long-term expression by fusing 6.3kb of upstream sequences from the rat tyrosine hydroxylase (TH) promoter to a NFH promoter, and this promoter has supported physiological studies. The TH promoter fragment contains an enhancer, as it has activity in both orientations and at a distance from the basal promoter. Identifying this enhancer may support further improvements in long-term expression. A previous deletion analysis identified two ~100bp fragments that each support long-term expression, and are contained within an ~320bp fragment located ~3kb from the TH promoter transcription start site. As this analysis used overlapping fragments, the two ~100bp fragments contained 44 or 23bp of unique sequence. Here, we used mutagenesis to identify a short sequence that supports long-term expression. We studied a 42bp sequence, centered on the 23bp unique sequence. Analysis of the wt sequence, and five mutations containing clustered changes that spanned the sequence, identified two adjacent mutations that do not support long-term expression, which together defined a 16bp maximum essential sequence. This 16bp sequence contains a putative E2F-1/DP-1 transcription factor binding site, and this transcription factor is expressed in many brain areas.

Evaluation of HSV-1 and adenovirus vector-mediated infection, replication and cytotoxicity in lymphoma cell lines

Lymphoma is not a common focus for viral therapy as many researchers do not consider lymphoma as a suitable target for viral vectors. In the present study, the infection, replication and cytotoxicity of herpes simplex virus (HSV) and adenovirus vectors were screened and evaluated in different lymphoma cell lines. Three recombinant viruses, BAC-HSV-1-eGFP, Adv-eGFP and an NV1020-like oncolytic HSV-1 virus strain named BAC-HSV-1-eGFP-delIRs, inserted with green fluorescent protein (GFP) as a reporter, were applied to evaluate the efficiency of viral infection and replication and cytotoxicity to lymphoma cells. Four types of lymphoma cell lines (SNK-6, Jurkat, Raji and Hut 78) were examined. We found that the HSV-1 vector, BAC-HSV-1-eGFP, was able to infect and replicate in the three lymphoma cell lines (Jurkat, Raji and Hut 78) and inhibit the growth of these cells more efficiently than adenovirus vector Adv-eGFP. The sensitivity of the four types of lymphoma cell lines to the viral vectors was different. The human cutaneous TL cell line Hut 78 was more sensitive to the viral vectors than the other cell lines. However, the human NK/T lymphoma cell line SNK-6 was not infected by any of the viral vectors and did not allow replication of the viruses. In conclusion, lymphoma may be a potential target for HSV-1 vector-mediated viral therapy.

Engineered Herpes Simplex Virus Vectors for Antitumor Therapy and Vaccine Delivery

Genetically modified herpes simplex viruses (HSVs) have been exploited for both antitumor therapy and vaccine delivery. These mutant viruses retain their ability to replicate and lyse permissive cells, including many tumor types, and are referred to as oncolytic HSVs. In addition, deletion of nonessential genes permits the introduction of foreign genes to augment the antitumor effect by either immune stimulation, targeting for select tumors, or expression of tumor or vaccine antigens. This article reviews the development of oncolytic HSVs as an anticancer therapy, as well as the application of HSV-1 vectors for delivery of targeted antigens or as vaccine adjuvants. The impact of these novel vectors with respect to enhanced antitumor activity and development of antitumor vaccination strategies is discussed.

Treatment of experimental autoimmune encephalomyelitis in SJL/J mice with a replicative HSV-1 vector expressing interleukin-5

Experimental autoimmune encephalomyelitis (EAE) is an autoimmune inflammation of the central nervous system and is used as the experimental model of multiple sclerosis (MS). The exact mechanism behind the disease is still unknown, but interleukin (IL)-17 expressing T cells are thought to mediate the disease. Toll-like receptors (TLRs) are known to have a role in the innate immune response against pathogens, and several TLRs have also a role in the disease course of EAE. Here, we show that treatment with a herpes simplex virus type 1 vector expressing the Th2 cytokine IL-5 ameliorates EAE and decreases the numbers of infiltrating lymphocytes in the brain. The effect involves downregulation of TLR 2, 3 and 9 mRNA expression and upregulation of type I interferons (IFNs) in brains during onset of disease. The elevated expression of type I IFNs was also observed during recovery.

Antibody-mediated targeted gene transfer to NMDA NR1-containing neurons in rat neocortex by helper virus-free HSV-1 vector particles containing a chimeric HSV-1 glycoprotein C–Staphylococcus A protein

Because of the heterogeneous cellular composition of the brain, and especially the forebrain, cell type-specific expression will benefit many potential applications of direct gene transfer. The two prevalent approaches for achieving cell type-specific expression are using a cell type-specific promoter or targeting gene transfer to a specific cell type. Targeted gene transfer with Herpes Simplex Virus (HSV-1) vectors modifies glycoprotein C (gC) to replace the heparin binding domain, which binds to many cell types, with a binding activity for a specific cell surface protein. We previously reported targeted gene transfer to nigrostriatal neurons using chimeric gC–glial cell line-derived neurotrophic factor or gC–brain-derived neurotrophic factor protein. Unfortunately, this approach is limited to cells that express the cognate receptor for either neurotrophic factor. Thus, a general strategy for targeting gene transfer to many different types of neurons is desirable. Antibody-mediated targeted gene transfer has been developed for targeting specific virus vectors to specific peripheral cell types; a specific vector particle protein is modified to contain the Staphylococcus A protein ZZ domain, which binds immunoglobulin (Ig) G. Here, we report antibody-mediated targeted gene transfer of HSV-1 vectors to a specific type of forebrain neuron. We constructed a chimeric gC–ZZ protein, and showed this protein is incorporated into vector particles and binds Ig G. Complexes of these vector particles and an antibody to the NMDA receptor NR1 subunit supported targeted gene transfer to NR1-containing neocortical neurons in the rat brain, with long-term (2months) expression.

Hot Topic: [Herpesvirus Vectors in Gene Therapy (Guest Editor: Veijo Hukkanen)

Herpes simplex virus type 1 (HSV-1) is a common human virus, best known as the causative agent of recurrent labial herpes (cold sores) [1]. The unique properties of HSV-1 have increasingly been exploited in vector design, particularly for therapy of the central nervous system diseases. HSV has natural properties useful for a therapy vector: the latent, life-long infections in the nervous system with long-term gene expression, the episomal non-integrating DNA genome and the numerous dispensable genes allowing cloning of large transgenes and their regulatory sequences [2]. Numerous clinical phase I-III studies using HSV vectors are in progress on therapy of gliomas and other malignancies and of other, mainly neurological, disorders, including pain [3, 4]. The HSV vectors can be genomic, nonreplicating or replicative vectors, or HSV amplicons. HSV amplicons are vectors harboring only minimal amount of virally derived sequences, suitable for delivery of up to 150 kbp of foreign DNA [5]. The use of HSV amplicon vectors has further been improved by the development of HSV-adeno-associated virus (AAV) hybrid vectors, aiming at long-term transgene expression. The wild type HSV, and also the HSV vectors, can evoke both innate and adaptive immune responses in the host organism [6]. The host responses can limit the vector persistence and duration of the transgene expression, but may occasionally also be advantageous in therapy of certain diseases. The current special issue introduces to the reader the different types and uses of HSV vectors, as well as the host responses which may affect the performance of these vectors. In this issue, Tsitoura and Epstein provide an expert overview of the HSV amplicon vectors and the host responses. They discuss the challenges in overcoming the amplicon silencing in the host, as well as the roles of certain HSV genes in counteracting the host responses. The review by de Oliveira and Fraefel introduces the HSV/AAV hybrid vectors, and illustrates also the biology of AAV and AAV vectors. The relevant helper viruses and HSV vectors are discussed, and new hybrid virus vectors are presented, such as HSV/Epstein-Barr virus and HSV/retrovirus hybrids. The overview by Cassady and Parker elucidates the use of HSV in tumor killing. They present the conditionally replicating HSV vectors for brain tumor therapy. In their paper, various approaches are illustrated for enhancement of tumor killing by HSV, such as combination of HSV virotherapy with other therapeutic modalities, or the tumor-specific targeting of HSV vectors. The authors discuss strategies to facilitate replication of HSV vectors in malignant cells without restoration of neurovirulence. In an extensive review, Manservigi et al. elucidate the design and applications of recombinant HSV vectors, with an emphasis on the genomic HSV vectors. They present the key issues of the HSV life cycle relevant to the vector development and introduce the different types of HSV vectors. The applications of HSV vectors are described in therapy of chronic diseases of the nervous system, in cancer therapy and also in vaccine vector design. The targeting strategies and means of imaging of HSV vectors are illustrated, and the authors also discuss the host responses and other factors compromizing the HSV vector efficacy. New perspectives for HSV-mediated tumor therapy are discussed, including the exploitation of microRNAs. This special issue of The Open Virology Journal presents state-of-the-art reviews by experts of the HSV vector field, covering the different types and uses of HSV vectors and elucidating the central questions such as vector design and targeting, silencing of the transgenes and other effects of the host responses. Hopefully this special issue is useful for the readership; for those interested in applications of HSV and for those considering the choice of viral vector for their particular gene therapy purpose.

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.

Herpesvirus Vectors for Therapy of Brain Tumors

Genetically modified, conditionally-replicating Herpes Simplex Virus Type 1 (HSV-1) vectors for the treatment of malignant glioma have provided encouraging results in the handful of Phase I and Phase II clinical trials conducted to date. In recent years, a number of new strategies have been developed to improve anti-tumor activity of these attenuated vectors, through either introduction of foreign gene inserts to enhance tumor killing through a variety of mechanisms, or through combination with existing treatment regimens, including radiation and/or chemotherapeutics. Another promising new approach has been the engineering of novel oncolytic HSV vectors that retain wildtype replication, but are targeted to tumor cells through a variety of mechanisms. This review summarizes the latest advances in herpesvirus-mediated oncolytic therapies from both preclinical results and clinical trials with oncolytic HSV vectors in patients, and their implication for design of future trials.

A helper virus-free HSV-1 vector containing the vesicular glutamate transporter-1 promoter supports expression preferentially in VGLUT1-containing glutamatergic neurons

Multiple potential uses of direct gene transfer into neurons require restricting expression to specific classes of glutamatergic neurons. Thus, it is desirable to develop vectors containing glutamatergic class-specific promoters. The three vesicular glutamate transporters (VGLUTs) are expressed in distinct populations of neurons, and VGLUT1 is the predominant VGLUT in the neocortex, hippocampus, and cerebellar cortex. We previously reported a plasmid (amplicon) Herpes Simplex Virus (HSV-1) vector that placed the Lac Z gene under the regulation of the VGLUT1 promoter (pVGLUT1lac). Using helper virus-free vector stocks, we showed that this vector supported ∼ 90% glutamatergic neuron-specific expression in postrhinal (POR) cortex, in rats sacrificed at either 4 days or 2 months after gene transfer. We now show that pVGLUT1lac supports expression preferentially in VGLUT1-containing glutamatergic neurons. pVGLUT1lac vector stock was injected into either POR cortex, which contains primarily VGLUT1-containing glutamatergic neurons, or into the ventral medial hypothalamus (VMH), which contains predominantly VGLUT2-containing glutamatergic neurons. Rats were sacrificed at 4 days after gene transfer, and the types of cells expressing ß-galactosidase were determined by immunofluorescent costaining. Cell counts showed that pVGLUT1lac supported expression in ∼ 10-fold more cells in POR cortex than in the VMH, whereas a control vector supported expression in similar numbers of cells in these two areas. Further, in POR cortex, pVGLUT1lac supported expression predominately in VGLUT1-containing neurons, and, in the VMH, pVGLUT1lac showed an ∼ 10-fold preference for the rare VGLUT1-containing neurons. VGLUT1-specific expression may benefit specific experiments on learning or specific gene therapy approaches, particularly in the neocortex.

Improved long-term expression from helper virus-free HSV-1 vectors packaged using combinations of mutated HSV-1 proteins that include the UL13 protein kinase and specific components of the VP16 transcriptional complex.

BackgroundHerpes Simplex Virus (HSV-1) gene expression is thought to shut off recombinant gene expression from HSV-1 vectors; however, in a helper virus-free HSV-1 vector system, a number of promoters support only short-term expression. These results raise the paradox that recombinant gene expression remains short-term even in the absence of almost all (~99%) of the HSV-1 genome, HSV-1 genes, and HSV-1 gene expression. To resolve this paradox, we hypothesized that specific proteins in the HSV-1 virus particle shut off recombinant gene expression. In two earlier studies, we examined the effects on recombinant gene expression of packaging vectors using specific mutated HSV-1 proteins. We found that vectors packaged using mutated UL13 (a protein kinase), or VP16, or UL46 and/or UL47 (components of the VP16 transcriptional complex) supported improved long-term expression, and vectors packaged using mutated UL46 and/or UL47 also supported improved gene transfer (numbers of cells at 4 days). These results suggested the hypothesis that specific proteins in the HSV-1 particle act by multiple pathways to reduce recombinant gene expression. To test this hypothesis, we examined combinations of mutated proteins that included both UL13 and specific components of the VP16 transcriptional complex.ResultsA HSV-1 vector containing a neuronal-specific promoter was packaged using specific combinations of mutated proteins, and the resulting vector stocks were tested in the rat striatum. For supporting long-term expression, the preferred combination of mutated HSV-1 proteins was mutated UL13, UL46, and UL47. Vectors packaged using this combination of mutated proteins supported a higher efficiency of gene transfer and high levels expression for 3 months, the longest time examined.ConclusionVector particles containing this combination of mutated HSV-1 proteins improve recombinant gene expression. Implications of these results for strategies to further improve long-term expression are discussed. Moreover, long-term expression will benefit specific gene therapy applications.

Oncolytic herpes simplex virus vectors and taxanes synergize to promote killing of prostate cancer cells

Genetically engineered oncolytic herpes simplex virus-1 (HSV-1) vectors selectively replicate in tumor cells causing direct killing whereas sparing normal cells. One clinical limitation of using oncolytic HSV vectors is their attenuated growth. We hypothesized that the appropriately chosen chemotherapeutic agent combined with an oncolytic HSV could be an effective means to promote augmented prostate cancer cell killing both in vitro and in vivo. Here we have identified that G47Delta synergizes with the microtubule-stabilizing taxane agents docetaxel and paclitaxel to enhance the in vitro killing of prostate cancer cells. In vivo efficacy studies show that when combined with docetaxel, G47Delta could be reduced at least 10-fold. Immunoblot analysis revealed that docetaxel-induced accumulation of the phospho-specific mitotic markers op18/stathmin or histone-H3 was markedly reduced by G47Delta, which correlated with enhanced apoptosis and required active viral replication. Furthermore, cell-cycle analysis demonstrated that in the presence of G47Delta, the majority of 4N cells arrested in mitosis were MPM-2-negative, indicative of cells exiting mitosis prematurely. These findings suggest that G47Delta may act in part, on mitotically blocked cells to enhance cell death, which may account for the enhanced antitumor efficacy observed in vivo.