Herpes Simplex Virus Amplicon Vector Research Articles

Development of herpes simplex virus-1 amplicon–based immunotherapy for chronic lymphocytic leukemia

Herpes simplex virus (HSV)-based vectors have favorable biologic features for gene therapy of leukemia and lymphoma. These include high transduction efficiency, ability to infect postmitotic cells, and large packaging capacity. The usefulness of HSV amplicon vectors for the transduction of primary human B-cell chronic lymphocytic leukemia (CLL) was explored. Vectors were constructed encoding beta-galactosidase (LacZ), CD80 (B7.1), or CD154 (CD40L) and were packaged using either a standard helper virus (HSVlac, HSVB7.1, and HSVCD40L) or a helper virus-free method (hf-HSVlac, hf-HSVB7.1, and hf-HSVCD40L). Both helper-containing and helper-free vector stocks were studied for their ability to transduce CLL cells, up-regulate costimulatory molecules, stimulate allogeneic T-cell proliferation in a mixed lymphocyte tumor reaction, and generate autologous cytotoxic T lymphocytes (CTLs). Although helper-containing and helper-free amplicon stocks were equivalent in their ability to transduce CLL cells, a vigorous T-cell proliferative response was obtained using cells transduced with hf-HSVB7.1 but not with HSVB7.1. CLL cells transduced with either HSVCD40L or hf-HSVCD40L were compared for their ability to up-regulate resident B7.1 and to function as T-cell stimulators. Significantly enhanced B7.1 expression in response to CD40L was observed using hf-HSVCD40L but not with HSVCD40L. CLL cells transduced with hf-HSVCD40L were also more effective at stimulating T-cell proliferation than those transduced with HSVCD40L stocks and were successful in stimulating autologous CTL activity. It is concluded that HSV amplicons are efficient vectors for gene therapy of hematologic malignancies and that helper virus-free HSV amplicon preparations are better suited for immunotherapy.

Gene therapy effectiveness differs for neuronal survival and behavioral performance.

If neuronal gene therapy is to be clinically useful, it is necessary to demonstrate neuroprotection when the gene is introduced after insult. We now report equivalent neuronal protection if calbindin D(28K) gene transfer via herpes simplex virus amplicon vector occurs immediately, 30 min, or 1 h after an excitotoxic insult, but not after a 4 h delay. Behavioral performance was evaluated for immediate and 1 h delay groups using a hippocampal-dependent task. Despite equivalent magnitude and pattern of sparing of neurons with the immediate and 1 h delay approaches, the delay animals took a significantly longer time after insult to return to normal performance.

A modular set of helper-dependent herpes simplex virus expression vectors.

Herpes simplex virus (HSV) has many favorable properties in terms of its potential to serve as a delivery and expression platform for gene-based therapies, including the ability to establish persistent infections, a broad tissue tropism, episomal maintenance of transduced genes, and a large genome that can incorporate many additional cDNAs. Helper-dependent HSV vectors (commonly known as HSV amplicons) are well positioned to exploit the biology of the virus, since they contain only the two cis elements required for HSV replication and packaging and thus do not require the silencing of any viral genes to prevent toxicity to transduced cells over the course of cDNA expression. In this report we describe the development of a set of modular HSV amplicon vectors that can easily be modified to incorporate different genetic elements or alternatively can be used to retrofit existing expression constructs such that they can be packaged into infectious HSV particles.

An adenoviral vector expressing the glucose transporter protects cultured striatal neurons from 3-nitropropionic acid

Considerable interest has focused on the possibility of using gene transfer techniques to introduce protective genes into neurons around the time of necrotic insults. We have previously used herpes simplex virus amplicon vectors to overexpress the rat brain glucose transporter, Glut-1 (GT), and have shown it to protect against a variety of necrotic insults both in vitro and in vivo, as well as to buffer neurons from the steps thought to mediate necrotic injury. It is critical to show the specificity of the effects of any such transgene overexpression, in order to show that protection arises from the transgene delivered, rather than from the vector delivery system itself. As such, we tested the protective potential of GT overexpression driven, in this case, by an adenoviral vector, against a novel insult, namely exposure of primary striatal cultures to the metabolic poison, 3-nitropropionic acid (3NP). We observed that GT overexpression buffered neurons from neurotoxicity induced by 3NP.

Herpes Simplex Virus (HSV)-Mediated ICAM-1 Gene Transfer Abrogates Tumorigenicity and Induces Anti-Tumor Immunity

Costimulatory and cellular adhesion molecules are thought to be essential components of antigen presentation in the immune response to cancer. The current studies examine gene transfer utilizing herpes viral amplicon vectors (HSV) to direct surface expression of adhesion molecules, and specifically evaluate the potential of a tumor-expressing intercellular adhesion molecule-1 (ICAM-1) to elicit an anti-tumor response. The human ICAM-1 (hICAM1) gene was inserted into an HSV amplicon vector and tested in a transplantable rat hepatocellular carcinoma and in a human colorectal cancer cell line. Cell surface ICAM-1 expression was assessed by flow cytometry. Lymphocyte binding to HSV-hICAM1-transduced cells was compared with that to cells transduced with HSV not carrying the ICAM gene. Tumorigenicity of HSV-hICAM1-transduced tumor cells were tested in syngeneic Buffalo rats. Additionally, immunization with irradiated (10,000 rads) HSV-hICAM1-transduced tumor cells was performed to determine its effect on tumor growth. A 20-min exposure of tumor cells at a multiplicity of infection (MOI) of 1 resulted in high-level cell surface expression of human ICAM in approximately 25% of tumor cells. Transduced rat or human tumor cells exhibited significantly enhanced binding of lymphocytes (p < 0.05). HSV-hICAM1-transduced cells elicited an increase in infiltration by CD4(+) lymphocytes in vivo and exhibited decreased tumorigenicity. Immunization with irradiated HSV-hICAM1-transduced cells protected against growth of subsequent injected parental tumor cells. HSV amplicon-mediated gene transfer is an efficient method for modifying the cell surface expression of adhesion molecules. Increased tumor expression of ICAM-1 represents a promising immune anti-cancer strategy.

Inhibition of NF2-negative and NF2-positive primary human meningioma cell proliferation by overexpression of merlin due to vector-mediated gene transfer

The absence of in vitro models of neurofibromatosis Type 2 (NF2)-defective meningiomas has limited investigative efforts to study the biological effects of this gene in the pathogenesis of these tumors. The goals of this report are to show that gene transfer vectors can efficiently express the wild-type NF2 transgene into primary meningioma cells and to determine effects on cellular proliferation. In this study, the authors have compared the transducing capacities of a retrovirus, an adenovirus, and a herpes simplex virus amplicon vector for use in primary human meningioma cells harvested from human tumors excised from patients with and without NF2. Transduction efficiencies with the latter vector approached 100% and it was selected to transfer the wild-type NF2 transgene into these cells. Western blot analysis confirmed that vector-mediated gene transfer mediated the expression of the NF2-encoded polypeptide merlin. Overexpression of merlin significantly inhibited the proliferation of both NF2-negative and NF2-positive human meningioma cells when compared to the proliferation of cells transduced with a control vector. This study demonstrates the feasibility of using vector-mediated gene transfer to study wild-type NF2 gene function in short-term cultures of primary human meningioma cells.

Eradication of Pre-Established Lymphoma Using Herpes Simplex Virus Amplicon Vectors

Herpes simplex virus amplicon vectors expressing RANTES (HSVrantes) and the T-cell costimulatory ligand B7.1 (HSVB7.1) were studied for their ability to elicit a tumor-specific T-cell response in a murine lymphoma model. HSVB7.1- and HSVrantes-transduced EL4 cells expressed high levels of B7.1 and RANTES as analyzed by flow cytometry and enzyme-linked immunosorbent assay, respectively. Inoculation of ex vivo HSVB7.1 transduced cells in syngeneic mice resulted in regression of both transduced cells and nontransduced cells inoculated contralaterally. Direct intratumoral injection of HSVB7.1 and/or HSVrantes alone or in combination into established EL4 tumors led to complete tumor regression in injected tumors as well as in nontransduced contralaterally implanted tumor, whereas control tumors or tumors injected with HSVlac expressing beta-galactosidase did not regress. Maximal protection was achieved with combined injection of HSVB7.1 and HSVrantes; mice showing tumor regression were resistant to rechallenge with parental EL4 cells, and tumor cell-specific cytolytic T-cell activity was observed in mice demonstrating regression. HSV amplicon-mediated delivery of immune effector molecules may represent a useful strategy for immunotherapy in the setting of pre-existing tumor.

Eradication of Pre-Established Lymphoma Using Herpes Simplex Virus Amplicon Vectors

AbstractHerpes simplex virus amplicon vectors expressing RANTES (HSVrantes) and the T-cell costimulatory ligand B7.1 (HSVB7.1) were studied for their ability to elicit a tumor-specific T-cell response in a murine lymphoma model. HSVB7.1- and HSVrantes-transduced EL4 cells expressed high levels of B7.1 and RANTES as analyzed by flow cytometry and enzyme-linked immunosorbent assay, respectively. Inoculation of ex vivo HSVB7.1 transduced cells in syngeneic mice resulted in regression of both transduced cells and nontransduced cells inoculated contralaterally. Direct intratumoral injection of HSVB7.1 and/or HSVrantes alone or in combination into established EL4 tumors led to complete tumor regression in injected tumors as well as in nontransduced contralaterally implanted tumor, whereas control tumors or tumors injected with HSVlac expressing β-galactosidase did not regress. Maximal protection was achieved with combined injection of HSVB7.1 and HSVrantes; mice showing tumor regression were resistant to rechallenge with parental EL4 cells, and tumor cell-specific cytolytic T-cell activity was observed in mice demonstrating regression. HSV amplicon-mediated delivery of immune effector molecules may represent a useful strategy for immunotherapy in the setting of pre-existing tumor.

Efficient gene transfer to human squamous cell carcinomas by the herpes simplex virus type 1 amplicon vector

Background: This study evaluates the efficiency of herpes simplex virus (HSV) mediated gene transfer in human squamous cell carcinoma (SCC) cell lines in vitro and in vivo when delivered by selective intra-arterial perfusion. Methods: Human head and neck SCC were exposed to HSV-LacZ and HSV–interleukin-2 (IL-2) and gene transfer and expression assessed by X-gal staining and enzyme-linked immunosorbent assay, respectively. Hamster cheek pouch tumors were perfused with HSV-LacZ or HSV–IL-2, by microcannulating the external carotid artery, and gene transfer determined. Results: A ratio of 5 viral particles per tumor cell achieved gene transfer rates exceeding 50%. Interleukin-2 levels of 287 ± 17 to 424 ± 8.4 ng per million cells were achieved at a ratio of 2 viral particles per tumor cell. Selective intra-arterial perfusion of the HSV–IL-2 vector yielded IL-2 levels of 45.8 ± 17.0 pg per g tumor. Conclusions: HSV amplicon vectors are efficient vehicles for gene transfer in vitro in human head and neck SCC cell lines and in vivo when introduced by selective intra-arterial perfusion.

Gene transfer of calbindin D28k cDNA via herpes simplex virus amplicon vector decreases cytoplasmic calcium ion response and enhances neuronal survival following glutamatergic challenge but not following cyanide.

Excitatory amino acid overstimulation of neurons can lead to a marked rise in cytoplasmic Ca2+ concentration ([Ca2+])i) and be followed by neuron death from hours to days later. If the rise in [Ca2+]i is prevented, either by removing Ca2+ from the extracellular environment or by placing Ca2+ chelators in the cytosol of the stimulated cells, the neurotoxicity associated with excitotoxins can be ameliorated. We have recently shown that neurons infected with a herpes simplex virus amplicon vector expressing cDNA for calbindin D28k responded to hypoglycemia with decreased [Ca2+]i and increased survival relative to controls. We now report that vector-infected neurons respond to glutamatergic insults with lower [Ca2+]i than controls and with increased survival. Infected neurons exposed to sodium cyanide did not respond with lower [Ca2+]i than controls, nor did they demonstrate increased survival postinsult. We examine these results in light of our earlier report and in the context of the potential of vectors like this for neuronal gene therapy.

An enhanced packaging system for helper-dependent herpes simplex virus vectors.

Helper-dependent herpes simplex virus (HSV) vectors (amplicons) show considerable promise to provide for long-term transduced-gene expression in most cell types. The current packaging system of choice for these vectors involves cotransfection with a set of five overlapping cosmids that encode the full HSV type 1 (HSV-1) helper virus genome from which the packaging (pac) elements have been deleted. Although both the helper virus and the HSV amplicon can replicate, only the latter is packaged into infectious viral particles. Since the titers obtained are too low for practical application, an enhanced second-generation packaging system was developed by modifying both the helper virus and the HSV amplicon vector. The helper virus was reverse engineered by using the original five cosmids to generate a single HSV-bacterial artificial chromosome (BAC) clone in Escherichia coli from which the pac elements were deleted to generate a replication-proficient but packaging-defective HSV-1 genome. The HSV amplicon was modified to contain the simian virus 40 origin of replication, which acts as an HSV-independent replicon to provide for the replicative expansion of the vector. The HSV amplicon is packaged into infectious particles by cotransfection with the HSV-BAC helper virus into the 293T cell line, and the resulting cell lysate is free of detectable helper virus contamination. The combination of both modifications to the original packaging system affords an eightfold increase in the packaged-vector yield.

Prolonged in vivo gene expression driven by a tyrosine hydroxylase promoter in a defective herpes simplex virus amplicon vector.

A 9.0-kb fragment of the tyrosine hydroxylase (TH) promoter, previously shown to direct tissue-specific expression in transgenic mice, was fused to an Escherichia coli LacZ reporter gene in a defective herpes simplex virus type-1 (HSV-1) amplicon vector (THlac). The HSV immediate early (IE) 4/5 promoter (HSVlac) was used as a control. LacZ gene expression was visualized by X-Gal histochemical and TH immunocytochemical analysis. Two days and 10 weeks after THlac injection into rat caudate nucleus (CN), X-Gal-stained cells were observed in the substantia nigra (SN) and locus ceruleus (LC) ipsilateral to the injection site. These blue cells were TH-positive neurons as evidenced by double labeling with immunocytochemistry. Moreover, the number of X-Gal+, TH+ (double-positive) neurons in the SN increased at 10 weeks as compared to that seen 2 days after THlac injection. In marked contrast, few double-positive nigral neurons were observed either 2 days or 10 weeks after direct injection of THlac into SN. However, neither nigral nor striatal injection of HSVlac resulted in prolonged gene expression. These results suggest that a neuronal, but not a viral, promoter in an HSV vector can produce cell-type-specific, prolonged, and stable gene expression following retrograde transport. In addition, THlac produced infrequent gene expression in TH-negative cells (CN and dorsal to SN) after THlac injection into CN and SN, respectively. Overall, these results suggest that in some in vivo contexts cell-type-preferred expression can be achieved by a cellular promoter in an amplicon vector. Moreover, they underscore the need for the careful and systematic study of neuronal promoters in HSV vectors.

Expression of the bcl-2 gene from a defective HSV-1 amplicon vector protects pancreatic beta-cells from apoptosis.

It has been suggested that the mechanism of pancreatic beta-cell death in autoimmune diabetes mellitus and in immunoisolated transplantation devices involves cytokine-induced apoptosis. To explore the feasibility of a gene transfer strategy to protect beta-cells, we evaluated the use of replication defective HSV-1 amplicon vectors as gene transfer vehicles. Post-mitotic murine and human beta-cells were efficiently transduced by a herpes simplex virus (HSV) vector that expresses the reporting gene Escherichia coli lacZ under the transcriptional control of a HSV promoter (HSVlac) both as islets and as single cells. Insulin secretion, a marker of beta-cell function, was unaffected by HSVlac transduction of a beta-cell line. A HSV amplicon vector that expressed bcl-2 (HSVbcl2) in beta-cells was constructed, and its effects on cytokine-mediated apoptosis in both a beta-cell line and primary murine beta-cells assessed by measuring internucleosomal fragmentation. beta-Cell apoptosis was blocked by transduction with HSVbcl2 but not HSVlac. The prevention of cytokine-induced apoptosis in beta-cells by bcl-2 expression has the potential both to ameliorate primary autoimmune beta-cell destruction as type I diabetes develops, and to prevent the destruction of transplanted beta-cells inside immunoisolation devices.