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1.
Gene Ther ; 17(3): 424-31, 2010 Mar.
Article in English | MEDLINE | ID: mdl-19865178

ABSTRACT

The ability of a viral vector to safely deliver and stably integrate large transgene units (transgenons), which not only include one or several therapeutic genes, but also requisite native transcriptional regulatory elements, would be of significant benefit for diseases presently refractory to available technologies. The herpes simplex virus type-1 (HSV-1) amplicon vector has the largest known payload capacity of approximately 130 kb, but its episomal maintenance within the transduced cell nucleus and induction of host cell silencing mechanisms limits the duration of the delivered therapeutic gene(s). Our laboratory developed an integration-competent version of the HSV-1 amplicon by adaptation of the Sleeping Beauty (SB) transposon system, which significantly extends transgene expression in vivo. The maximum size limit of the amplicon-vectored transposable element remains unknown, but previously published plasmid-centric studies have established that DNA segments longer than 6-kb are inefficiently transposed. Here, we compared the transposition efficiency of SB transposase in the context of both the HSV amplicon vector as well as the HSV amplicon plasmid harboring 7 and 12-kb transposable reporter transgene units. Our results indicate that the transposition efficiency of the 12-kb transposable unit via SB transposase was significantly reduced as compared with the 7-kb transposable unit when the plasmid version of the HSV amplicon was used. However, the packaged HSV amplicon vector form provided a more amenable platform from which the 12-kb transposable unit was mobilized at efficiency similar to that of the 7-kb transposable unit via the SB transposase. Overall, our results indicate that SB is competent in stably integrating transgenon units of at least 12 kb in size within the human genome upon delivery of the platform via HSV amplicons.


Subject(s)
DNA Transposable Elements/genetics , Gene Transfer Techniques , Genetic Therapy/methods , Genetic Vectors , Herpesvirus 1, Human/genetics , Transposases/genetics , Virus Integration , HeLa Cells , Humans , Transgenes
2.
J Biomed Mater Res A ; 87(1): 116-28, 2008 Oct.
Article in English | MEDLINE | ID: mdl-18085643

ABSTRACT

A range of substrates made of polystyrene (PS) and poly(methyl methacrylate)-poly(methacrylic acid) copolymer (PMMA-PMAA) containing 98 and 80% PMMA (PA98, PA80) and presenting a homogeneous or a patterned surface were used to study fibronectin adsorption and neuronal cell behavior. Fibronectin adsorption showed weak differences regarding the adsorbed amount (evaluated by XPS), but large differences in adsorbed layer morphology as observed by AFM. A fine granular morphology, with dimensions up to 8 nm height and 50-150 nm width, was observed on top of a thin adsorbed layer in the case of PS, PA98, and of a surface made of nanoscale inclusions of the latter in PS. In contrast, the layer adsorbed on PA80, which carries more ionizable groups, showed a higher roughness on the PA80 zones with differences in height up to 30 nm and characteristic lateral dimensions of 400 nm. On substrates of the former category, the cells formed large clusters, revealing poor interactions with the substrate. On PA80, the cells formed large networks with only a few small clusters. The adsorbed layer roughness, resulting from aggregation of fibronectin upon adsorption and from the substrate surface chemical composition, is responsible for neuronal cell spreading and growth. Its effect is not prevented by the presence of inclusions (< 30% of the surface) responsible for smoother areas of adsorbed fibronectin and for protrusions below 40 nm.


Subject(s)
Fibronectins/chemistry , Neurons/cytology , Polymethacrylic Acids/chemistry , Polystyrenes/chemistry , Adsorption , Animals , Brain , Cell Adhesion , Cells, Cultured , Embryo, Mammalian , Fibronectins/ultrastructure , Mice , Microscopy, Atomic Force , Microscopy, Fluorescence , Neurons/metabolism , Substrate Specificity , Surface Properties
3.
Neurobiol Aging ; 29(1): 71-7, 2008 Jan.
Article in English | MEDLINE | ID: mdl-17101197

ABSTRACT

Alzheimer's disease (AD) is a common and devastating neurodegenerative disease in which most cases are of unknown, sporadic origin. In addition to age, the most prevalent known risk factor for developing AD is carriage of the epsilon4 allele of Apolipoprotein E (ApoE). Carriage of the epsilon2 or epsilon3 allele of ApoE confers protection or no change in risk for AD, respectively. Latent herpes simplex virus type 1 (HSV-1) infection in the brain concurrent with ApoE4 carriage exacerbates risk for AD, suggesting that these two factors interact to promote neuronal dysfunction and degeneration in selective brain areas. Indeed, HSV-1 DNA has been found in regions primarily affected by AD, such as the temporal lobes, hippocampus, and neocortex. We hypothesize that HSV-1 infection in the background of ApoE4, but not ApoE2 or ApoE3, promotes an environment more conducive to neuronal degeneration. To investigate this idea, we have utilized transgenic mice that express human ApoE2, 3, or 4 alleles from astrocytes in a murine ApoE -/- background. We find that carriage of the different ApoE alleles dramatically affects HSV-1 immediate early gene expression as well as the establishment of latency. Both of these factors are poised to impact neuronal viability, inflammation, and viral spread. Our data support the concept that HSV-1 and ApoE4 interact to provide an environment conducive to the development and/or spread of AD.


Subject(s)
Apolipoproteins E/metabolism , Genes, Immediate-Early/physiology , Herpesvirus 1, Human/physiology , Neurons/virology , Virus Latency/physiology , Age Factors , Animals , Apolipoproteins E/deficiency , Cells, Cultured , Cornea/virology , Embryo, Mammalian , Female , Gene Expression Regulation, Viral/physiology , Humans , Male , Mice , Mice, Inbred C57BL , Mice, Transgenic , Neurons/pathology , Transduction, Genetic/methods
4.
J Gene Med ; 8(11): 1320-8, 2006 Nov.
Article in English | MEDLINE | ID: mdl-16989006

ABSTRACT

Given their generous transgene capacity and inherent neurotropism, herpes simplex virus (HSV-1)-based viral vectors are promising tools for gene delivery to the central nervous system. Despite their widespread pre-clinical use, vector toxicity remains a concern with regard to the use of herpes vectors in humans. One potential source of toxicity stems from the tegument-associated virion host shutoff protein (vhs), which induces translational arrest in the host cell through non-specific mRNAse activity. In the current study we utilized a series of HSV-1 viruses containing a deletion in the U(L)41 open reading frame to investigate: (1) the requirement of intact vhs function in amplicon packaging and (2) whether vhs influences the post-transduction survival of dissociated cortical neurons. Our results demonstrate that while amplicon yield was reduced an order of magnitude, U(L)41 deletion was associated with reduced vector toxicity. Furthermore, partial reconstitution of vhs function using mRNAse-inactive point mutants improved amplicon titers without imparting the toxicity observed with wild-type controls. These findings offer a novel approach to improving the titer and toxicity profiles of HSV-based viral vectors.


Subject(s)
Herpesvirus 1, Human/genetics , Animals , Cell Line , Cell Survival , Chlorocebus aethiops , Genes, Viral , Herpesvirus 1, Human/physiology , In Vitro Techniques , Mice , Mice, Inbred C57BL , Mutation , NIH 3T3 Cells , Neurons/cytology , Neurons/virology , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA, Viral/genetics , RNA, Viral/metabolism , Ribonucleases/genetics , Ribonucleases/metabolism , Sequence Deletion , Vero Cells , Viral Proteins/genetics , Viral Proteins/metabolism , Virus Assembly
5.
Genesis ; 44(1): 44-9, 2006 Jan.
Article in English | MEDLINE | ID: mdl-16419044

ABSTRACT

The cre/LoxP system can produce conditional loss of gene function in specific cell types such as neurons. A transgenic mouse line, utilized by multiple studies, used the Synapsin I promoter to drive expression of cre (SynCre) to achieve neuronal-specific cre expression. Herein we describe that cre expression can also be observed in SynCre mice within the testes after being bred into a floxed transgenic mouse line. Cre transcript was expressed in testes resulting in recombination of the floxed substrate in testes. In the majority of cases, progeny of male SynCre mice inherited a germline recombined floxed allele, while this was never observed in progeny from female mice carrying the SynCre allele. This observation should alert investigators to a potential confound using these mice and enables male germ cell "deletor" strategies.


Subject(s)
Germ Cells , Integrases/genetics , Recombination, Genetic , Synapsins/genetics , Transgenes , Animals , Base Sequence , DNA Primers , Male , Mice , Mice, Transgenic , Neurons/cytology , Neurons/metabolism , Polymerase Chain Reaction , Testis/metabolism
6.
Article in English | MEDLINE | ID: mdl-16315613

ABSTRACT

The convergent pathobiologic model of Parkinson's disease stipulates that disparate insults initiate a disease process that obligately share a common pathway leading to cell death. A combinatorial treatment which targets various steps in this pathway is likely to be the most successful therapeutic strategy. As advances are made in the field of neuroimaging and pharmacogenomics, early detection of sporadic PD will become a reality. Early intervention will likely spare more dopaminergic neurons and extend the quality of life for the patient. Continued advancements in the fields of pharmacology, neurosurgery, and gene therapy will strengthen the armamentarium available for the treatment of PD patients.


Subject(s)
Genetic Therapy/methods , Neurodegenerative Diseases/therapy , Animals , Antioxidants/metabolism , Dopamine/metabolism , Gene Transfer Techniques , Humans , Mutation , Neurodegenerative Diseases/genetics , Neurons/pathology , Neuroprotective Agents/pharmacology , Parkinson Disease/genetics , Parkinson Disease/therapy
7.
Neurobiol Aging ; 26(5): 765-75, 2005 May.
Article in English | MEDLINE | ID: mdl-15708451

ABSTRACT

The gradual loss of striatal dopamine and dopaminergic neurons residing in the substantia nigra (SN) causes parkinsonism characterized by slow, halting movements, rigidity, and resting tremor when neuronal loss exceeds a threshold of approximately 80%. It is estimated that there is extensive compensation for several years prior to symptom onset, during which vulnerable neurons asynchronously die. Recent evidence would argue that much of the compensatory response of the nigrostriatal system is multimodal including both pre-synaptic and striatal mechanisms. Although parkinsonism may have multiple causes, the classic syndrome, Parkinson's disease (PD), is frequently modeled in small animals by repeated administration of the selective neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Because the MPTP model of PD recapitulates many of the known behavioral and pathological features of human PD, we asked whether the striatal cells of mice treated with MPTP in a semi-chronic paradigm enact a transcriptional program that would help elucidate the response to dopamine denervation. Our findings reveal a time-dependent dysregulation in the striatum of a set of genes whose products may impact both the viability and ability to communicate of dopamine neurons in the SN.


Subject(s)
1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine/pharmacology , Corpus Striatum/drug effects , Gene Expression/drug effects , MPTP Poisoning/metabolism , Analysis of Variance , Animals , Corpus Striatum/metabolism , Disease Models, Animal , MPTP Poisoning/genetics , Male , Mice , Mice, Inbred C57BL , Models, Biological , Oligonucleotide Array Sequence Analysis/methods , Principal Component Analysis/methods , Reproducibility of Results
9.
J Alzheimers Dis ; 6(6 Suppl): S43-6, 2004 Dec.
Article in English | MEDLINE | ID: mdl-15665413

ABSTRACT

Herein a case is made for the development of novel cytoprotective approaches based upon molecular mechanisms thought to underlie the caloric restriction phenomenon. This analysis leads to the prediction that molecular genetic perturbations affecting the metabolism of nuclear NAD(+) and metabolites will be neuroprotective.


Subject(s)
Alzheimer Disease , Amide Synthases/genetics , Amide Synthases/metabolism , Caloric Restriction/methods , Energy Intake , Genetic Therapy/methods , Alzheimer Disease/enzymology , Alzheimer Disease/genetics , Alzheimer Disease/prevention & control , Animals , Cerebral Cortex/enzymology , Drosophila , Feeding Behavior , Gene Transfer Techniques/instrumentation , Neurons/enzymology , Oxidative Stress/physiology , Risk Factors
10.
Exp Neurol ; 183(1): 47-55, 2003 Sep.
Article in English | MEDLINE | ID: mdl-12957487

ABSTRACT

Direct intracerebral administration of glial cell line-derived neurotrophic factor (GDNF) is neuroprotective against ischemia-induced cerebral injury. Utilizing viral vectors to deliver and express therapeutic genes presents an opportunity to produce GDNF within localized regions of an evolving infarct. We investigated whether a herpes simplex virus (HSV) amplicon-based vector encoding GDNF (HSVgdnf) would protect neurons against ischemic injury. In primary cortical cultures HSVgdnf reduced oxidant-induced injury compared to the control vector HSVlac. To test protective effects in vivo, HSVgdnf or HSVlac was injected into the cerebral cortex 4 days prior to, or 3 days, after a 60-min unilateral occlusion of the middle cerebral artery. Control stroke animals developed bradykinesia and motor asymmetry; pretreatment with HSVgdnf significantly reduced such motor deficits. Animals receiving HSVlac or HSVgdnf after the ischemic insult did not exhibit any behavioral improvement. Histological analyses performed 1 month after stroke revealed a reduction in ischemic tissue loss in rats pretreated with HSVgdnf. Similarly, these animals exhibited less immunostaining for glial fibrillary acidic protein and the apoptotic marker caspase-3. Taken together, our data indicate that HSVgdnf pretreatment provides protection against cerebral ischemia and supports the utilization of the HSV amplicon for therapeutic delivery of trophic factors to the CNS.


Subject(s)
Genetic Vectors/administration & dosage , Ischemic Attack, Transient/prevention & control , Nerve Growth Factors/administration & dosage , Nerve Growth Factors/genetics , Simplexvirus/genetics , Animals , Astrocytes/drug effects , Astrocytes/metabolism , Astrocytes/pathology , Brain/blood supply , Brain/drug effects , Brain/pathology , Caspase 3 , Caspases/metabolism , Cells, Cultured , Disease Models, Animal , Genetic Therapy/methods , Genetic Vectors/genetics , Glial Cell Line-Derived Neurotrophic Factor , Glial Fibrillary Acidic Protein/metabolism , Hydrogen Peroxide/toxicity , Immunohistochemistry , Ischemic Attack, Transient/pathology , Motor Activity/drug effects , Nerve Growth Factors/biosynthesis , Neurons/drug effects , Neurons/metabolism , Neurons/pathology , Neuroprotective Agents/administration & dosage , Oxidants/toxicity , Rats , Rats, Sprague-Dawley , Recovery of Function , Treatment Outcome
12.
Gene Ther ; 10(11): 941-5, 2003 Jun.
Article in English | MEDLINE | ID: mdl-12756414

ABSTRACT

Herpes simplex virus (HSV) is a naturally occurring double-stranded DNA virus that has been adapted into an efficient vector for in vivo gene transfer. HSV-based vectors exhibit wide tropism, large transgene size capacity, and moderately prolonged transgene expression profiles. Clinical implementation of HSV vector-based gene therapy for prevention and/or amelioration of human diseases eventually will be realized, but inherently this goal presents a series of significant challenges, one of which relates to issues of immune system involvement. Few experimental reports have detailed HSV vector-engendered immune responses and subsequent resolution events primarily within the confines of the central nervous system. Herein, we describe the immunobiology of HSV and its derived vector platforms, thus providing an initiation point from where to propose requisite experimental investigation and potential approaches to prevent and/or counter adverse antivector immune responses.


Subject(s)
Central Nervous System/immunology , Central Nervous System/virology , Genetic Therapy/adverse effects , Genetic Vectors/immunology , Herpesvirus 1, Human/immunology , Virus Diseases/immunology , Animals , Antibodies, Viral/immunology , Complement Inactivator Proteins/immunology , Genetic Engineering/methods , Humans , Recombinant Proteins/immunology , Viral Envelope Proteins/immunology , Virus Replication/physiology
13.
Curr Opin Mol Ther ; 3(5): 482-90, 2001 Oct.
Article in English | MEDLINE | ID: mdl-11699893

ABSTRACT

The efficient and targeted transfer of genes is the goal of gene therapy. In the central nervous system (CNS), this is challenging due in part to the exquisite anatomy of the brain. Herpes simplex virus (HSV) vectors are particularly amenable to CNS therapies as they are capable of transducing a variety of cells, have a large transgene capacity and can exist as either oncolytic or non-immunogenic vectors. The versatility of this vector platform and its potential molecular therapeutic use in two CNS disorders, Alzheimer's disease and malignant brain tumors, will be discussed.


Subject(s)
Central Nervous System Diseases/therapy , Genetic Therapy/methods , Genetic Vectors , Simplexvirus/genetics , Alzheimer Disease/therapy , Amyloid beta-Peptides/genetics , Amyloid beta-Peptides/immunology , Animals , Brain Neoplasms/therapy , Central Nervous System/metabolism , Combined Modality Therapy , Gene Transfer Techniques , Genetic Therapy/trends , Humans , Prodrugs/therapeutic use , Vaccines, Synthetic/therapeutic use
14.
Hum Gene Ther ; 12(15): 1867-79, 2001 Oct 10.
Article in English | MEDLINE | ID: mdl-11589829

ABSTRACT

There is currently much interest in generating cytotoxic T lymphocyte (CTL) responses against tumor antigens as a therapy for cancer. This work describes a novel gene transfer technique utilizing dendritic cells (DCs), an extremely potent form of antigen-presenting cell (APC), and herpes simplex virus-1 (HSV-1) amplicons. HSV-1 amplicons are plasmid-based viral vectors that are packaged into HSV-1 capsids, but lack viral coding sequences. Amplicon vectors have been constructed that encode the model tumor antigen ovalbumin (HSV-OVA) and human prostate-specific antigen (HSV-PSA), a protein that is expressed specifically in prostate epithelium and prostate carcinoma cells. These amplicons were packaged using a helper virus-free system that produces vector stocks that are devoid of contaminating cytotoxic helper virus. Transduction of DCs with HSV-OVA or HSV-PSA and co-culture with CTL hybridomas results in specific activation, indicating that transduced DCs express these transgenes and process the tumor antigens for class I MHC presentation to CTL. Mice immunized with HSV-PSA-transduced DCs generate a specific CTL response that can be detected in vitro by a (51)Cr-release assay and are protected from challenge with tumors that express PSA. These results indicate that DCs transduced with HSV-1 amplicon vectors may provide a tool for investigation of the biology of CTL activation by DCs and a new modality for immunotherapy of cancer.


Subject(s)
Cancer Vaccines , Dendritic Cells/cytology , Dendritic Cells/metabolism , Herpesvirus 1, Human/genetics , Immunotherapy/methods , Neoplasms/therapy , Prostate-Specific Antigen/biosynthesis , Animals , Antigen-Presenting Cells/metabolism , Cell Line , Chromium Radioisotopes/metabolism , Coculture Techniques , Epithelial Cells/metabolism , Flow Cytometry , Genetic Vectors , Green Fluorescent Proteins , Humans , Hybridomas , Luminescent Proteins/metabolism , Male , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Plasmids/metabolism , Prostatic Neoplasms/metabolism , Time Factors , Transduction, Genetic
15.
Mol Ther ; 4(3): 250-6, 2001 Sep.
Article in English | MEDLINE | ID: mdl-11545616

ABSTRACT

There are two promising herpes viral-based anticancer strategies: one involves replication-defective viruses to transfer therapeutic transgenes, and the other involves replication-conditional oncolytic viruses, which selectively infect and destroy cancer cells directly. This study examines a novel dual herpesvirus preparation, which combines the immunostimulatory effects of amplicon-mediated IL2 expression with direct viral-induced oncolysis. The oncolytic virus G207 was used as the helper virus to package a herpes simplex virus (HSV)-amplicon vector carrying the gene IL2 (HSV-IL2), yielding a single preparation with two complementary modes of action. In vivo comparison was carried out in a syngeneic squamous cell carcinoma flank tumor model. We directly injected established tumors with HSV-IL2, G207, G207 mixed with HSV-IL2, or G207-packaged HSV-amplicon carrying the IL2 transgene (G207[IL2]). Significant inhibition of tumor growth was seen at 2 weeks in the G207[IL2]-treated tumors relative to controls (0.57+/-0.44 cm(3) versus 39.45+/-5.13 cm(3), P<0.00001), HSV-IL2 (20.97+/-4.60 cm(3)), and the G207 group (7.71+/-2.10 cm(3)). This unique use of a replication-conditional, oncolytic virus to package a replication-incompetent amplicon vector demonstrates impressive efficacy in vitro and in vivo, and avoids the theoretical concerns of recombination with reversion to wild type.


Subject(s)
Immunotherapy , Interleukin-2/genetics , Interleukin-2/immunology , Neoplasms/genetics , Neoplasms/therapy , Simplexvirus/genetics , Animals , CD4-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/immunology , Carcinoma, Squamous Cell/genetics , Carcinoma, Squamous Cell/immunology , Carcinoma, Squamous Cell/pathology , Carcinoma, Squamous Cell/therapy , Cell Death , Cell Division , Humans , Interleukin-2/metabolism , Interleukin-2/therapeutic use , Mice , Neoplasms/immunology , Neoplasms/pathology , Transfection , Tumor Cells, Cultured
16.
Drug Metab Dispos ; 29(10): 1256-62, 2001 Oct.
Article in English | MEDLINE | ID: mdl-11560867

ABSTRACT

Evidence for the presence of a novel transporter in primary cultures of rat striatal neurons and mouse cortical neurons similar in function to the multidrug resistance-associated protein (MRP1) is presented. Functional activity was assessed by efflux studies with the glutathione conjugate of monochlorobimane (B-SG). The glutathione transferase-catalyzed formation of B-SG in rat striatal neurons and mouse cortical neurons was inhibited by ethacrynic acid. The efflux of B-SG from rat striatal neurons and mouse cortical neurons was lower at 20 degrees C than at 37 degrees C and was lower in cells with reduced ATP concentrations compared with cells with constitutive ATP concentrations. In addition, the efflux of B-SG was inhibited by MK-571 in both rat striatal and mouse cortical neurons and by probenecid in rat striatal neurons, but not in mouse cortical neurons. Verapamil did not inhibit B-SG efflux in either rat striatal or mouse cortical neurons. Although functionally similar to MRP1, Western blot analysis with commercially available antibodies directed against human and mouse MRP1 failed to show MRP1-like protein in either whole-cell homogenates of rat striatal neurons or mouse cortical neurons, indicating that the described neuronal transporter differs in structure from human or mouse MRP1 or lacks epitopes in common with MRP1.


Subject(s)
ATP-Binding Cassette Transporters/metabolism , Glutathione/metabolism , Neurons/metabolism , Pyrazoles/metabolism , ATP-Binding Cassette Transporters/antagonists & inhibitors , ATP-Binding Cassette Transporters/isolation & purification , Adenosine Triphosphate/metabolism , Animals , Biological Transport , Blotting, Western , Cells, Cultured , Glutathione/chemistry , Mice , Multidrug Resistance-Associated Proteins , Pyrazoles/chemistry , Rats , Temperature , Time Factors , Visual Cortex/cytology
17.
Neuron ; 31(3): 353-65, 2001 Aug 16.
Article in English | MEDLINE | ID: mdl-11516394

ABSTRACT

The transcriptional repressor, REST, helps restrict neuronal traits to neurons by blocking their expression in nonneuronal cells. To examine the repercussions of REST expression in neurons, we generated a neuronal cell line that expresses REST conditionally. REST expression inhibited differentiation by nerve growth factor, suppressing both sodium current and neurite growth. A novel corepressor complex, CoREST/HDAC2, was shown to be required for REST repression. In the presence of REST, the CoREST/HDAC2 complex occupied the native Nav1.2 sodium channel gene in chromatin. In neuronal cells that lack REST and express sodium channels, the corepressor complex was not present on the gene. Collectively, these studies define a novel HDAC complex that is recruited by the C-terminal repressor domain of REST to actively repress genes essential to the neuronal phenotype.


Subject(s)
Cerebral Cortex/physiology , Neurons/physiology , Repressor Proteins/metabolism , Transcription Factors/metabolism , Animals , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , COS Cells , Cell Differentiation/drug effects , Cell Line , Cells, Cultured , Chlorocebus aethiops , Chromatin/physiology , DNA-Binding Proteins/metabolism , Embryo, Mammalian , Histone Deacetylase 2 , Histone Deacetylases/genetics , Histone Deacetylases/metabolism , Humans , Mice , Mice, Inbred C57BL , NAV1.2 Voltage-Gated Sodium Channel , Nerve Growth Factor/pharmacology , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/physiology , Neurons/cytology , Neurons/drug effects , PC12 Cells , Rats , Recombinant Fusion Proteins/metabolism , Recombinant Proteins/metabolism , Repressor Proteins/genetics , Serine Endopeptidases/genetics , Serine Endopeptidases/metabolism , Sodium Channels/genetics , Sodium Channels/physiology , Transcription Factors/genetics , Transfection , Zinc Fingers
18.
Blood ; 98(2): 287-95, 2001 Jul 15.
Article in English | MEDLINE | ID: mdl-11435295

ABSTRACT

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.


Subject(s)
Genetic Vectors , Herpesvirus 1, Human/genetics , Immunotherapy , Leukemia, Lymphocytic, Chronic, B-Cell/immunology , Leukemia, Lymphocytic, Chronic, B-Cell/therapy , Antigen-Presenting Cells , B7-1 Antigen/genetics , CD40 Ligand/genetics , Cytokines/metabolism , Histocompatibility Antigens Class I/genetics , Histocompatibility Antigens Class I/immunology , Humans , Lymphocyte Activation , T-Lymphocytes/immunology , T-Lymphocytes, Cytotoxic/immunology , Transfection , beta-Galactosidase/genetics
19.
J Virol ; 75(15): 7050-8, 2001 Aug.
Article in English | MEDLINE | ID: mdl-11435585

ABSTRACT

G207 is an oncolytic herpes simplex virus (HSV) which is attenuated by inactivation of viral ribonucleotide reductase (RR) and deletion of both gamma(1)34.5 genes. The cellular counterparts that can functionally substitute for viral RR and the carboxyl-terminal domain of ICP34.5 are cellular RR and the corresponding homologous domain of the growth arrest and DNA damage protein 34 (GADD34), respectively. Because the thymidylate synthetase (TS) inhibitor fluorodeoxyuridine (FUdR) can alter expression of cellular RR and GADD34, we examined the effect of FUdR on G207 bioactivity with the hypothesis that FUdR-induced cellular changes will alter viral proliferation and cytotoxicity. Replication of wild-type HSV-1 was impaired in the presence of 10 nM FUdR, whereas G207 demonstrated increased replication under the same conditions. Combined use of FUdR and G207 resulted in synergistic cytotoxicity. FUdR exposure caused elevation of RR activity at 10 and 100 nM, whereas GADD34 was induced only at 100 nM. The effect of enhanced viral replication by FUdR was suppressed by hydroxyurea, a known inhibitor of RR. These results demonstrate that the growth advantage of G207 in FUdR-treated cells is primarily based on an RR-dependent mechanism. Although our findings show that TS inhibition impairs viral replication, the FUdR-induced RR elevation may overcome this disadvantage, resulting in enhanced replication of G207. These data provide the cellular basis for the combined use of RR-negative HSV mutants and TS inhibitors in the treatment of cancer.


Subject(s)
Antiviral Agents/pharmacology , Floxuridine/pharmacology , Herpesvirus 1, Human/enzymology , Ribonucleotide Reductases/metabolism , Viral Proteins/metabolism , Virus Replication/drug effects , Animals , Antigens, Differentiation , Cell Cycle , Cell Cycle Proteins , Chlorocebus aethiops , Gene Expression , Genes, Reporter , Herpesvirus 1, Human/drug effects , Herpesvirus 1, Human/physiology , Humans , Mutagenesis, Insertional , Protein Phosphatase 1 , Proteins/genetics , Ribonucleotide Reductases/genetics , Tumor Cells, Cultured , Vero Cells , Viral Proteins/genetics , beta-Galactosidase/genetics
20.
Mol Ther ; 3(6): 958-63, 2001 Jun.
Article in English | MEDLINE | ID: mdl-11407910

ABSTRACT

Ototoxicity is a major dose-limiting side effect of cisplatin (DDP) administration due to its propensity to induce destruction of hair cells and neurons in the auditory system. Previous studies demonstrated that TrkC-expressing spiral ganglion neurons (SGN) are protected from the cytotoxic effects of DDP by localized delivery of the trophic factor neurotrophin-3 (NT-3). Successful in vivo implementation of such a therapy requires the development of an efficient gene delivery vehicle for expression of NT-3 within the cochlea. To this end, we constructed a herpes simplex virus (HSV) amplicon vector that expressed a c-Myc-tagged NT-3 chimera (HSVnt-3myc). Helper virus-free vector stocks were initially evaluated in vitro for their capacity to direct expression of NT-3 mRNA and protein. Transduction of cultured murine cochlear explants with HSVnt-3myc resulted in production of NT-3 mRNA and protein up to 3 ng/ml as measured over a 48-h period in culture supernatants. To determine whether NT-3 overexpression could abrogate DDP toxicity, cochlear explants were transduced with HSVnt-3myc or a murine intestinal alkaline phosphatase-expressing control vector, HSVmiap, and then exposed to cisplatin. HSVnt-3myc-transduced cochlear explants harbored significantly greater numbers of surviving SGNs than those infected with control virus. These data demonstrate that amplicon-mediated NT-3 transduction can attenuate the ototoxic action of DDP on organotypic culture. The potency of NT-3 in protecting spiral ganglion neurons from degeneration suggests that in vivo neurotrophin-based gene therapy may be useful for the prevention and/or treatment of hearing disorders.


Subject(s)
Cisplatin/toxicity , Neurotrophin 3/metabolism , Simplexvirus/genetics , Spiral Ganglion/drug effects , Animals , Blotting, Western , Cell Death/drug effects , Cochlea/metabolism , Cytomegalovirus/genetics , DNA Primers/chemistry , Enzyme-Linked Immunosorbent Assay , Genes, myc/genetics , Genes, myc/physiology , Hearing Disorders/chemically induced , Hearing Disorders/prevention & control , Immunoenzyme Techniques , Neurites/chemistry , Neurons/drug effects , Neurons/pathology , Neurotrophin 3/genetics , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Spiral Ganglion/pathology
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