Herpes Simplex Virus Amplicon Research Articles

Adoptively transferred tumor-specific T cells expanded ex vivo using HSV amplicons encoding 4-1BBL persist in the host and demonstrate anti-tumor activity in vivo. (88.11)

Abstract 4-1BB is a T cell costimulatory receptor which promotes T cell expansion and survival. We studied the anti-tumor effects of adoptively transferred tumor-specific T cells expanded ex vivo using herpes simplex virus (HSV) amplicons encoding either 4-1BBL (HSV.4-1BBL) or B7.1 (HSV.B7). Lewis Lung Carcinoma cells expressing ovalbumin (LLC/OVA) were transduced with HSV.4-1BBL, HSV.B7, or control HSV and used to stimulate GFP+ OVA-specific CD8+ T cells (OT-1). Naïve or ex vivo-stimulated OT-1 were then transferred into LLC/OVA-bearing mice. Mice given 4-1BBL-stimulated OT-1 had significantly decreased tumor volumes compared to untreated mice (P<0.0001) or mice given naïve OT-1 (P=0.0003). Transfer of B7-stimulated OT-1 did not control tumor growth. Higher percentages of OT-1 were seen in the blood, draining lymph nodes, and tumor for the 4-1BBL-stimulated OT-1 group compared to other groups at 8 days post transfer. BrdU incorporation of 4-1BBL-stimulated OT-1 was markedly increased within the tumor bed. Splenocytes from the 4-1BBL-stimulated OT-1 group showed increased cytolytic activity against LLC/OVA and higher numbers of OT-1 cells, which expressed CD44 and the memory marker Ly-6C, compared to controls. Tumor-specific T cells stimulated ex vivo using tumor transduced with HSV.4-1BBL expand efficiently in vivo resulting in tumor eradication and the generation of tumor-specific memory T cells.

Effects of ex vivo transduction of mesencephalic reaggregates with bcl-2 on grafted dopamine neuron survival

Survival rates of dopamine (DA) neurons grafted to the denervated striatum are extremely poor (5–20%). Gene transfer of survival promoting factors, such as the anti-apoptotic protein bcl-2, to mesencephalic DA neurons prior to transplantation (ex vivo transduction) offers a novel approach to increase graft survival. However, specific criteria to assess the efficacy of various vectors must be adhered to in order to reasonably predict successful gene transfer with appropriate timing and levels of protein expression. Cell culture results utilizing three different herpes simplex virus (HSV) vectors to deliver the reporter β-galactosidase gene (lacZ) indicate that transduction of mesencephalic cells with a helper virus-free HSV amplicon (HF HSV-TH9lac) that harbors the 9-kb tyrosine hydroxylase (TH) promoter to drive lacZ gene expression elicits the transduction of the highest percentage (≈50%) of TH-immunoreactive (THir) neurons without significant cytotoxic effects. This transduction efficiency and limited cytotoxicity was superior to that observed following transduction with helper virus-containing HSV (HC HSVlac) and helper virus-free HSV amplicons (HF HSVlac) expressing lacZ under the transcriptional control of the HSV immediate-early 4/5 gene promoter. Subsequently, we assessed the ability of HSV-TH9lac and the bcl-2 expressing HSV-TH9bcl-2 amplicon to transduce mesencephalic reaggregates. Although an increase in bcl-2 and β-galactosidase protein was induced by transduction, amplicon-mediated overexpression of bcl-2 did not lead to an increase in grafted THir neuron number. Even with highly efficient viral vector-mediated transduction, our results demonstrate that ex vivo gene transfer of bcl-2 to mesencephalic reaggregates is ineffective in increasing grafted DA neuron survival.

Synergy between expression of fusogenic membrane proteins, chemotherapy and facultative virotherapy in colorectal cancer

Using Chou-Talalay median effect analysis, we demonstrated in permanent and short-term cultures of colorectal cancer cells that the expression of measles virus fusogenic membrane glycoproteins (FMGs) in combination with chemotherapy often causes over most of the cytotoxic dose range synergistic cell killing. In this combined treatment, we observed strongly enhanced annexin V binding and caspase-3/7 activity when compared to single-agent treatment. Furthermore, we showed increased expression of heat-shock protein (Hsp)70 and Hsp90alpha, but not of Hsp60. In a subcutaneous HT-29 colorectal xenograft model, we demonstrated that the administration of a replication-defective adenoviral or herpes simplex virus (HSV) amplicon vector (Ad.H/F or HSV.H/F) encoding tumor-restricted FMG in combination with FOLFOX significantly enhanced treatment outcome when compared to treatment with each compound individually. To increase the fraction of tumor cells expressing the FMG, we trans-complemented the Ad.H/F and HSV.H/F vector with the respective oncolytic replication-restricted adenovirus Ad.COXDeltaMK or HSV-1 G47Delta vector. At the end of the observation period (day 100), eight out of 10 animals that received G47Delta, HSV.H/F and FOLFOX were alive and tumor free. Administration of the analogous adenovirus-based regimen resulted in four out of 10 long-term survivors. We demonstrated that the expression of FMG in combination with chemotherapy can significantly enhance treatment outcome, which is further enhanced by combination with trans-complementing oncolytic vectors.

DNA-Based Methods to Prepare Helper Virus-Free Herpes Amplicon Vectors and Versatile Design of Amplicon Vector Plasmids

The herpes simplex virus (HSV) amplicon vector is a versatile plasmid-based gene delivery vehicle with a large transgene capacity (up to 150 kb) and the ability to infect a broad range of cell types. The vector system was originally developed by Frenkel and her colleagues in 1980. Ever since, a great deal of effort by various investigators has been directed at minimizing the toxicity associated with the inevitable contamination by helper virus. In 1996, Fraefel and his colleagues successfully devised a cosmid-based packaging system that was free of contamination by helper virus (so-called helper virus-free packaging), which utilized as helper a set of 5 overlapping cosmid clones that covered the entire HSV genome, which lacked the DNA packaging/cleavage signals. With the helper virus-free system, broader applications of the vector became possible. Cloning of the entire HSV genome in bacteria artificial chromosome (BAC) plasmids enabled stable maintenance and propagation of the helper HSV genome in bacteria. It also allowed for the development of BAC-based helper virus-free packaging systems. In this article, we review various versions of DNA-based methods to prepare HSV amplicon vectors free of helper virus contamination. We also examine recent advances in vector design, including methods of vector construction, hybrid amplicon vectors, and the infectious BAC system. Future directions in improving packaging systems and vector designs are discussed.

Gene therapy in the nervous system with superoxide dismutase

Neuronal death following necrotic insults involves the generation of reactive oxygen species (ROS). We investigated the effects of antioxidant gene therapy on ROS accumulation after exposure to either sodium cyanide, kainic acid or oxygen glucose deprivation (OGD). Specifically, we generated herpes simplex virus-1 amplicon vector expressing the gene for the antioxidant enzyme CuZnSOD. Overexpression of this gene in primary hippocampal cultures resulted in increased enzymatic activity of the corresponding protein. CuZnSOD significantly protected hippocampal neurons against sodium cyanide insult and the subsequent lipid peroxidation. However, it did not protect against OGD- or kainic-acid-induced toxicity. Moreover, CuZnSOD significantly worsened the toxicity, hydrogen peroxide accumulation and lipid peroxidation induced by kainic acid. As a possible explanation for this surprising worsening, CuZnSOD overexpression increased glutathione peroxidase activity in the presence of sodium cyanide but had no effect on catalase or glutathione peroxidase activity in the presence of kainic acid. Thus, cells were unlikely to be able to detoxify the excess hydrogen peroxide produced as a result of the CuZnSOD overexpression. These studies can be viewed as a cautionary note concerning gene therapy intervention against necrotic insults.

Plasmid DNA Sequences Present in Conventional Herpes Simplex Virus Amplicon Vectors Cause Rapid Transgene Silencing by Forming Inactive Chromatin

The herpes simplex virus (HSV)-based amplicon vector, a bacterial-viral-mammalian cell shuttle system, holds promise as a versatile gene delivery vehicle because of its large transgene capacity. However, amplicon-mediated transgene expression is often transient. We hypothesized that the presence of prokaryotic DNA sequences within the packaged vector genome can trigger transcriptional silencing of the entire vector sequence. To test this, we constructed a novel amplicon vector devoid of bacterial sequences (minicircle [MC] amplicon). Although the same dose of the minicircle amplicon vector in normal human fibroblasts resulted in an expression of luciferase approximately 20 times higher than that caused by the conventional amplicon vector, no significant difference was observed in copy numbers of luciferase DNA between MC amplicon- and control-transduced cells. Quantitative analyses of levels of luciferase mRNA revealed that differential expression of luciferase was controlled at the transcriptional level. Chromatin immunoprecipitation PCR analyses of several regions of vector genomes revealed that the bacterial sequences found in the conventional amplicon DNA were associated with an inactive form of chromatin immediately after infection. The presence of bacterial sequences also affected the remaining vector sequences in the conventional amplicon vector. Finally, nude mice injected with the MC amplicon exhibited higher and more sustained expression of luciferase than those injected with the conventional amplicon, confirming the usefulness of the MC amplicon devoid of bacterial sequences. Although additional improvements are absolutely required, these findings are a significant first step toward developing a novel HSV amplicon vector that can achieve enhanced long-term transgene expression.

158. Real-Time Visualization of Neuronal Differentiation and Neurite Projection Using an Indicator HSV Amplicon Vector

Top of pageAbstract Stem cell-based replacement therapy has great potential in treating neurological disorders, including brain and spinal cord injuries. Although several studies have demonstrated neuronal and glial differentiation of transplanted neural stem cells (NSCs) in vivo, the efficacy of the transplanted NSCs to generate neurons and of the newly generated neurons to form new synaptic connections with the remaining host neurons still needs clarification. To address these issues, we have constructed an HSV amplicon-based indicator vector that allows visualization of neuronal differentiation and neurite projection. Synapsin I (SYN) is a well characterized gene that expresses selectively in neurons. Thiel and colleagues found that a 400-bp 5' untranslated promoter region of SYN directed prominent expression of a reporter gene in various neuronal cell lines, but that no expression was detected in cell lines derived from non-neuronal tissue (Thiel G et al., PNAS 1991). On the other hand, microtubule- associated protein tau-reporter fusion protein has been used as a genetic reporter to visualize neuronal processes in vivo (Callahan CA et al., PNAS 1994). We, therefore, combined the two genetic elements in the context of HSV amplicon vectors, producing an indicator herpes simplex virus (HSV) amplicon vector, HSV-SYN-tauR, that carries tau fused with a monomeric red fluorescent protein (mRFP, Campbell et al., PNAS 2002) under the control of the 400- bp SYN promoter. When a mixed culture of mouse cortical neurons and glia was transduced with the HSV-SYN-tauR amplicon vector, RFP reporter was expressed only in neurons along their neurites, allowing visualization of every axon, dendrite, and spine. Time-lapse imaging of mixed cortical cultures transduced with the HSV-SYN-tauR amplicon vector allowed noninvasive real-time monitoring of extension and retraction of the neurites of live neurons. This indicator HSV amplicon vector could prove useful in studying neuronal differentiation, neurite projections, and regeneration of neuronal networks in vitro and in vivo.

156. Rapid Methods of Engineering HSV Amplicon Vectors Kazue Kasai, Yoshinaga Saeki

The herpes simplex virus (HSV) amplicon vector is a versatile plasmid-based gene delivery vehicle. Its unique features include a transgene capacity up to 150 kb, the ability to transduce a broad range of mammalian cell types, and the availability of a truly helper virus-free packaging system. These characteristics make the vector system attractive for gene therapy and basic biological research. The basic structure of an HSV amplicon vector, an amplicon vector plasmid, comprises a plasmid backbone, two cis-acting, non-coding viral sequences (an origin of DNA replication and a DNA cleavage/ packaging signal), and one or more transgene cassettes of interest. When an amplicon vector plasmid is transfected into HSV-permissive cells (e.g., Vero cells) and supplied with HSV helper functions in trans, the amplicon plasmid DNA undergoes a |[ldquo]|head-to-tail|[rdquo]| rolling circular-type DNA replication, and the resulting concatenated structures of the original amplicon plasmid can be packaged into an infectious HSV virion as a 150-kb linear, double-stranded DNA molecule.

1068. Plasmid DNA Sequences Present in Conventional HSV Amplicon Vectors Cause and Rapid Transgene Silencing by Inactive Chromatin Formation

The HSV-based amplicon vector, a bacteria-virus-mammalian cell shuttle system, holds promise as a versatile gene delivery vehicle because of its large transgene capacity. However, amplicon-mediated transgene expression is often transient. We hypothesized that the presence of prokaryotic DNA sequences within the packaged vector genome can trigger transcriptional silencing of the entire vector sequence. To test this, we constructed a novel amplicon vector devoid of bacterial sequences (minicircle amplicon). Although the same dose of the minicircle (MC) amplicon vector in normal human fibroblasts resulted in expression of luciferase approximately 20 times higher than that produced by the conventional amplicon vector, no significant difference was observed in copy numbers of luciferase DNA between MC amplicon- and control-transduced cells. Quantitative analyses of levels of luciferase mRNA revealed that differential expression of luciferase was controlled at the transcriptional level. Chromatin immunoprecipitation PCR analyses of several regions of vector genomes revealed that the bacterial sequences found in the conventional amplicon DNA were associated with an inactive form of chromatin immediately after infection. The presence of bacterial sequences also affected the remaining vector sequences in the conventional amplicon vector. Similar results were obtained with nondividing normal human astrocytes in culture, precluding the possibility that this phenomenon is fibroblast specific. Finally, nude mice injected with MC amplicon exhibited higher and more sustained expression of luciferase than those injected with conventional amplicon, confirming the usefulness of the MC amplicon devoid of bacterial sequences. These findings are a significant first step towards developing a novel herpes simplex virus (HSV) amplicon vector that can achieve enhanced long-term transgene expression.

154. Tetracycline-Inducible HSV Amplicon Vectors with Expanded Range of Gene Expression Regulation

In this study, we developed several novel tetracycline(tet)-regulated Herpes Simplex Virus-1 (HSV) amplicon vectors by introducing various combinations of tet-dependent transcriptional silencers and activators. Also, to reduce undesirable baseline gene expression, most vectors incorporated insulator elements flanking the gene expression cassette, which carried the Firefly luciferase gene (FLuc) driven by a silencer/activator-responsive promoter. To determine which constructs were most effective at regulating gene expression, we tested the luciferase activity of transduced human glioblastoma Gli-36 cells in vitro, in the presence or absence of doxycycline (Dox). Induction levels for the twenty-six vectors tested ranged from 154-fold to 6000-fold, with the best vector (HET14B-FLuc) displaying over 5-fold higher luciferase activity than a control HSV amplicon vector constituitively expressing luciferase. Dose response analysis showed that gene expression could be regulated over a wide range of Dox concentrations, and that 1000-fold induction could be achieved at 10 ng/mL Dox. To further demonstrate the regulation capabilities of this system, we replaced the luciferase gene in HET14B-FLuc with the gene for reversed caspase-3, an auto-catalytic version of wild-type caspase-3, an executioner of apoptosis. WST cell proliferation assays demonstrate that Gli-36 cells transduced with this vector (MOI 0.5-2) undergo rapid apoptosis upon Dox induction, with almost no viable cells remaining 6 hours post-induction. Non-induced cells infected with the same vector, as well as induced and non-induced cells infected with HET14BFLuc control vector, continued growing throughout the 36 hour assay. Application of the pan-caspase inhibitor Z-VAD-FMK at the time of induction prevented apoptosis in cells infected with the caspase-3 vector, and cell proliferation levels were similar to those in control groups. Western blot analysis, probing for poly-(ADP-ribose)-polymerase, which is cleaved during apoptosis, confirm that cells infected with caspase-3 vector undergo massive apoptosis upon Dox induction, while control groups exhibit a low baseline level of cell death in all samples. Currently we are evaluating the therapeutic potential of the caspase-3 vector in vivo by direct injection into subcutaneous Gli-36 tumors constitutively expressing Fluc. Subsequent tumor growth in the presence or absence of Dox will be assessed by bioluminescence imaging of tumor- associated FLuc.

Ryanodine Receptor Type 1 (RyR1) Mutations C4958S and C4961S Reveal Excitation-coupled Calcium Entry (ECCE) Is Independent of Sarcoplasmic Reticulum Store Depletion

Bi-directional signaling between ryanodine receptor type 1 (RyR1) and dihydropyridine receptor (DHPR) in skeletal muscle serves as a prominent example of conformational coupling. Evidence for a physiological mechanism that upon depolarization of myotubes tightly couples three calcium channels, DHPR, RyR1, and a Ca(2+) entry channel with SOCC-like properties, has recently been presented. This form of conformational coupling, termed excitation-coupled calcium entry (ECCE) is triggered by the alpha(1s)-DHPR voltage sensor and is highly dependent on RyR1 conformation. In this report, we substitute RyR1 cysteines 4958 or 4961 within the TXCFICG motif, common to all ER/SR Ca(2+) channels, with serine. When expressed in skeletal myotubes, C4958S- and C4961S-RyR1 properly target and restore L-type current via the DHPR. However, these mutants do not respond to RyR activators and do not support skeletal type EC coupling. Nonetheless, depolarization of cells expressing C4958S- or C4961S-RyR1 triggers calcium entry via ECCE that resembles that for wild-type RyR1, except for substantially slowed inactivation and deactivation kinetics. ECCE in these cells is completely independent of store depletion, displays a cation selectivity of Ca(2+)>Sr(2+) approximately Ba(2+), and is fully inhibited by SKF-96365 or 2-APB. Mutation of other non-CXXC motif cysteines within the RyR1 transmembrane assembly (C3635S, C4876S, and C4882S) did not replicate the phenotype observed with C4958S- and C4961S-RyR1. This study demonstrates the essential role of Cys(4958) and Cys(4961) within an invariant CXXC motif for stabilizing conformations of RyR1 that influence both its function as a release channel and its interaction with ECCE channels.

Herpes Simplex Virus Amplicon Delivery of a Hypoxia-Inducible Soluble Vascular Endothelial Growth Factor Receptor (sFlk-1) Inhibits Angiogenesis and Tumor Growth in Pancreatic Adenocarcinoma

Tumor hypoxia induces vascular endothelial growth factor (VEGF) expression, which stimulates angiogenesis and tumor proliferation. The VEGF signaling pathway is inhibited by soluble VEGF receptors (soluble fetal liver kinase 1; sFlk-1), which bind VEGF and block its interaction with endothelial cells. Herpes simplex virus (HSV) amplicons are replication-incompetent viruses used for gene delivery. We attempted to attenuate angiogenesis and inhibit pancreatic tumor growth through HSV amplicon-mediated expression of sFlk-1 under hypoxic control. A multimerized hypoxia-responsive enhancer (10 x HRE) was cloned upstream of the sFlk-1 gene (10 x HRE/sFlk-1). A novel HSV amplicon expressing 10 x HRE/sFlk-1 was genetically engineered (HSV10 x HRE/sFlk-1).Human pancreatic adenocarcinoma cells (AsPC1) were transduced with HSV10 x HRE/sFlk-1 and incubated in normoxia (21% oxygen) or hypoxia (1% oxygen). Capillary inhibition was evaluated by human umbilical vein endothelial cell assay. Western blot assessed sFlk-1 expression. AsPC1 flank tumor xenografts (n = 24) were transduced with HSV10 x HRE/sFlk-1. Media from normoxic AsPC1 transduced with HSV10 x HRE/sFlk-1 yielded a 36% reduction in capillary formation versus controls (P < .05), whereas hypoxic AsPC1 yielded a 76% reduction (P < .005). Western blot of AsPC1 transduced with HSV10 x HRE/sFlk-1 demonstrated greater sFlk-1 expression in hypoxia versus normoxia. AsPC1 flank tumors treated with HSV10 x HRE/sFlk-1 exhibited a 59% reduction in volume versus controls (P < .000001). HSV amplicon delivery of a hypoxia-inducible soluble VEGF receptor significantly reduces new vessel formation and tumor growth. Tumor hypoxia can thus be used to direct antiangiogenic therapy to pancreatic adenocarcinoma.

Use of Herpes Virus Amplicon Vectors to Study Brain Disorders

There is an enormous initiative to establish the genetic basis for disorders of brain function. Unfortunately, genetic intervention is not accomplished easily in the nervous system. One strategy is to engineer and deliver to neurons specialized viral vectors that carry a gene (or genes) of interest, thereby exploiting the natural ability of viruses to insert genetic material into cells. When delivered to brain cells, these vectors cause infected cells to increase the expression of the genes of interest. The ability to deliver genes into neurons in vitro and in vivo with herpes simplex virus (HSV) amplicon vectors has made it possible to carry out exactly these sorts of experiments. This technology has the potential to offer new insights into the etiology of a wide variety of neuropsychiatric disorders. We describe the use of HSV amplicon vectors to study Alzheimer disease, drug addiction, and depression, and discuss the considerations that enter into the use of these vectors both in vitro and in vivo. The HSV amplicon virus is a user-friendly vector for the delivery of genes into neurons that has come of age for the study of brain function.

Human Dendritic Cells Transduced with Herpes Simplex Virus Amplicons Encoding Human Immunodeficiency Virus Type 1 (HIV-1) gp120 Elicit Adaptive Immune Responses from Human Cells Engrafted into NOD/SCID Mice and Confer Partial Protection against HIV-1 Challenge

Small-animal models are needed to test human immunodeficiency virus (HIV) vaccine efficacy following viral challenge. To this end, we examined HIV-1-specific immune responses following immunization of nonobese diabetic-severe combined immunodeficient mice that were repopulated with human peripheral blood lymphocytes (hu-PBL-NOD/SCID mice). Autologous dendritic cells (DC) were transduced ex vivo with replication-defective, helper virus-free, herpes simplex virus type 1 (HSV-1) amplicons that expressed HIV-1 gp120 and were then injected into the hu-PBL-NOD/SCID mice. This resulted in primary HIV-1-specific humoral and cellular immune responses. Serum samples from vaccinated animals contained human immunoglobulin G that reacted with HIV-1 Env proteins by enzyme-linked immunosorbent assay and neutralized the infectivity of HIV-1 LAI and ADA strains. T cells isolated from the mice responded to viral antigens by producing gamma interferon when analyzed by enzyme-linked immunospot assay. Importantly, exposure of the vaccinated animals to infectious HIV-1 demonstrated partial protection against infectious HIV-1 challenge. This was reflected by a reduction in HIV-1(ADA) and by protection of the engrafted human CD4(+) T lymphocytes against HIV-1(LAI)-induced cytotoxicity. These data demonstrate that transduction of DC by HSV amplicon vectors expressing HIV-1 gp120 induce virus-specific immune responses in hu-PBL-NOD/SCID mice. This mouse model may be a useful tool to evaluate human immune responses and protection against viral infection following vaccination.

Human cytomegalovirus plasmid-based amplicon vector system for gene therapy

We have constructed and evaluated the utility of a helper-dependent virus vector system that is derived from Human Cytomegalovirus (HCMV). This vector is based on the herpes simplex virus (HSV) amplicon system and contains the HCMV orthologs of the two cis-acting functions required for replication and packaging of HSV genomes, the complex HCMV viral DNA replication origin (oriLyt), and the cleavage packaging signal (the a sequence). The HCMV amplicon vector replicated independently and was packaged into infectious virions in the presence of helper virus. This vector is capable of delivering and expressing foreign genes in infected cells including progenitor cells such as human CD34+ cells. Packaged defective viral genomes were passaged serially in fibroblasts and could be detected at passage 3; however, the copy number appeared to diminish upon serial passage. The HCMV amplicon offers an alternative vector strategy useful for gene(s) delivery to cells of the hematopoietic lineage.

Herpes simplex virus amplicon delivery of a hypoxia-inducible angiogenic inhibitor blocks capillary formation in hepatocellular carcinoma

Tumor hypoxia induces vascular endothelial growth factor (VEGF) expression, which stimulates tumor angiogenesis. The VEGF pathway is inhibited by soluble VEGF receptors (soluble fetal liver kinase-1 [sFlk-1]) that bind VEGF and block its interaction with endothelial cells. Herpes simplex virus (HSV)-derived amplicons are replication-incompetent viruses used for gene delivery. We attempt to attenuate angiogenesis and inhibit hepatoma growth through amplicon-mediated expression of sFlk-1 under hypoxic control. A multimerized hypoxia-responsive enhancer (10xHRE) was cloned upstream of the sFlk-1 gene (10xHRE/sFlk-1). An amplicon expressing 10xHRE/sFlk-1 was genetically engineered (HSV10xHRE/sFlk-1). SK-HEP-1 human hepatoma cells were transduced with HSV10xHRE/sFlk-1 and incubated in normoxia (21% O2) or hypoxia (1% O2). Human umbilical vein endothelial cell assay evaluated capillary inhibition. Western blot assessed sFlk-1 expression. SK-HEP-1 flank tumors (n = 24) in athymic mice were treated with HSV10xHRE/sFlk-1. Media from hypoxic SK-HEP-1 transduced with HSV10xHRE/sFlk-1 yielded an 80% reduction in capillary formation (P < 0.005), whereas normoxic SK-HEP-1 yielded a 25% reduction (P < 0.05). Western blot of SK-HEP-1 transduced with HSV10xHRE/sFlk-1 demonstrated greater sFlk-1 expression in hypoxia vs. normoxia. SK-HEP-1 tumors treated with HSV10xHRE/sFlk-1 yielded a 72% reduction in volume vs. the control group (P < 0.000001). HSV amplicon-mediated delivery of a hypoxia-inducible soluble VEGF receptor substantially reduces new vessel formation and tumor growth in hepatoma.

GnRH is expressed in GnRH-deficient hypogonadal (hpg) mice after GnRH gene transfer with a herpes simplex virus (HSV) amplicon vector

The challenge of gene therapy is to develop non-toxic vectors that can achieve stable transgene expression in target cells. The HSV amplicon is a unique vector that has essentially no toxicity, a large transgene capacity, and the potential to integrate the transgene into a specific site in the genome, thus making it a useful tool for gene therapy. Hpg mice are genetically deficient in GnRH production in the hypothalamus, resulting in many deficits in the reproductive system including infertility. Our goal was to determine neuronal expression of GnRH and the recovery of gonadal function after CNS delivery of an HSV amplicon vector containing the GnRH gene using hpg mice. An HSV amplicon containing the GnRH gene under its cognate promoter was prepared and packaged using a helper virus-free packaging system. This vector was injected into the preoptic area (POA) of the hypothalamus of hpg mice using a stereotaxic apparatus. Following injection, we determined GnRH neuronal expression by immunohistochemistry (IHC) and recovery of gonadal function using vaginal cytology and blood hormonal assays. Eight adult female hpg mice were studied. Five mice were injected with the HSV-GnRH vector in the POA, and 3 mice were injected with vehicle only as control. The HSV-GnRH vector (1× 108 transducing units/ml) was injected into the POA of hpg mice under anesthesia using a stereotaxic apparatus. Mice were sacrificed 10 days (n=4) or 50 days (n=1) after injection. Neuronal expression of GnRH was analyzed by IHC in brain tissue. Plasma FSH and LH levels were measured by radioimmunoassay. The weights of the ovary, uterus and oviduct were compared between the study and control groups. Vaginal cytology was also checked each day after HSV amplicon injection to evaluate estrous cyclicity. GnRH expression was present in neurons near the injection site, as determined by IHC 10 days and 50 days following the vector injections. However, there was no significant difference in plasma FSH (2.0 ± 0.7 ng/ml vs. 4.9 ± 0.9 ng/ml; study vs. control), LH (0.07 ± 0.01 ng/ml vs. 0.06 ± 0.02 ng/ml) levels, or in the weight of reproductive organs (24.2 ± 6.4 mg vs. 17.9 ± 2.7 mg) between the study and control groups. We observed only diestrus specific morphology in the vaginal epithelial cells. Expression of GnRH was observed in the hypothalamic neurons in the POA in GnRH-deficient hpg mice following GnRH gene transfer using an HSV amplicon vector. These data suggest that such HSV vectors may be useful in correcting the reproductive defect in these animals. Further studies are necessary to determine if normal hypothalamic-pituitary-gonadal axis function can be restored in hpg mice by gene therapy using this approach.

HSV-1 amplicon-mediated transfer of 128-kb BMP-2 genomic locus stimulates osteoblast differentiation in vitro

In previous studies, we developed mouse genetic models and discovered genetic components of quantitative trait loci on mouse chromosomes that contribute to phenotypes such as bone size, bone density, and fracture healing. However, these regions contain dozens of genes in several overlapping bacterial artificial chromosomes (BACs) and are difficult to clone by physical cloning strategies. A feasible and efficient approach of identifying candidate genes is to transfer the genomic loci in BAC clones into mammalian cells for functional studies. In this study, we retrofitted a BAC construct into herpes simplex virus-1 amplicon and packaged it into an infectious BAC (iBAC) to test gene function in a cell-based system, using a 128-kb clone containing the complete bone morphogenetic protein-2 (BMP-2) gene. We transduced MC3T3-E1 cells with the iBAC bearing BMP-2 gene and examined transgene expression and function. Our results have demonstrated that an iBAC can efficiently deliver a BMP-2 genomic locus into preosteoblast cells and express functional BMP-2 protein, inducing a phenotype of cell differentiation, as indicated by an increase in alkaline phosphatase activity. Therefore, this experimental system provides a rapid, efficient cell-based model of high-throughput phenotypic screening to identify the BAC clones from physically mapped regions that are important for osteoblast differentiation. It also illustrates the potential of iBAC technology in functional testing of single nucleotide polymorphisms located in the distal promoter or/and intron regions responsible for low bone density.

211. Creation of a HSV VP22-Sleeping Beauty Fusion Protein To Facilitate Integration of HSV-1 Amplicon Vectors

One major challenge facing gene delivery using the Herpes simplex virus (HSV) amplicon system is its inability to impart long-term gene expression within post-mitotic cells of the CNS. Integration of the transgene into an active chromosomal region of the host cell genome may facilitate longer term gene expression profiles. Such an advance would bring together the benefits of HSV amplicon gene delivery including neurotropism and large transgene capacity with those of integrating virus vector systems such as adeno-associated virus and lentiviruses, which have been shown to express transgene products for protracted periods, on the order of months/years. We have previously employed an HSV amplicon vector-adapted form of the Tc1-like Sleeping Beauty (SB) transposase system to extend transgene expression in mitotically active and post-mitotic cells. Co-transduction of an RSV promoter driven β-galactosidase-neomycin (βgeo) fusion transgene flanked by the SB inverted/direct repeats (HSVT-βgeo) with an amplicon expressing Sleeping Beauty (HSVsb) in BHK cells, murine primary neurons, and adult mouse striata in vivo resulted in integration and extended transgene expression. A disadvantage with this approach lies in the amplicon-based delivery of the SB gene, which could result in catalysis of numerous integration events and potential deleterious cellular effects. To minimize this outcome, a bacterially expressed version of the Sleeping Beauty transposase was developed that can be mixed with amplicon virions encoding a transposable substrate to provide a transient burst of integration activity. We have fused VP22, a transcytotic HSV-1 structural protein that is efficiently imported into mammalian cells and localized to the nucleus, to the Sleeping Beauty transposase. HSVT-βgeo was used to infect BHK cells in combination with varying amounts of purified VP22-Sleeping Beauty transposase fusion protein. Using drug-resistant colony formation as a functional assay, our results demonstrate that the VP22-Sleeping Beauty protein is able to enter cells and catalyze integration of an HSV amplicon-harbored transposable element. Further studies are in progress, including in vivo assessment of this integrating vector system. This advancement is an exciting development in HSV amplicon-mediated gene delivery and promises to expand the utility of this vector platform.

The Potential of Gene Transfer into Primary B-CLL Cells Using Recombinant Virus Vectors

Despite recent advances, chronic lymphocytic leukemia (CLL) as the most common leukemia remains a largely incurable disease. Modern treatment options include novel drugs like purine analogues, monoclonal antibodies and transplantation strategies. Moreover, gene transfer of immunostimulatory molecules is another, but still experimental approach that can be used to potentiate immune responses against leukemic cells. CD40 ligand (CD40L) was shown to be a promising molecule for immunotherapy of B-CLL playing a critical role in immune activation. However, CLL B cells are resistant to transduction with most currently available vector systems. Improving the efficiency and specificity of gene vectors is critical for the success of gene therapy in this area. Using replication defective adenovirus encoding CD40L (Ad-CD40L), immunologic and clinical responses were seen in CLL patients after infusion of autologous Ad-CD40L-CLL cells in a recent phase I trial. Due to the immunogenic nature of adenovirus vectors, alternative vector systems are currently explored. Recombinant adeno-associated virus (rAAV) was shown to enable efficient transduction of primary B-CLL cells. By use of a library of AAV clones with randomly modified capsids, receptor-targeting mutants with a tropism for CLL cells can be selected. Furthermore, helper-virus free Epstein-Barr virus (EBV)-based gene transfer vectors hold promise for development of CLL-targeted vaccines after remaining safety issues will be resolved. Herpes simplex virus (HSV)-based vectors, especially HSV amplicons, have favorable features for B-CLL gene transfer including high transduction efficiency, ability to infect postmitotic cells and a large packaging capacity. The challenge for the future will be to transfer these alternative vector systems into clinic and allow the detection of a CLL-specific immune response by use of defined tumor antigens. This will make it possible to establish the potential clinical role of gene therapy for CLL patients.