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Biomedical subjects

J C Glorioso

Publications and source records attributed to J C Glorioso.

At least 19 recordsLinked to original sources
PubMed2000

Cytokine therapy in immune-mediated demyelinating diseases of the central nervous system: a novel gene therapy approach.

Pro-inflammatory cytokines play a crucial role in the regulatory and effector phase of the immune-mediated mechanism sustaining multiple sclerosis pathogenesis (MS) thus supporting the use of anti-inflammatory cytokines as a therapeutic option. Systemic administration of cytokines shows, however, limited therapeutic efficacy and undesirable/unpredictable side-effects. We have developed a non-toxic system to deliver cytokines within the central nervous system (CNS) based on the intrathecal (i.c.) administration of non-replicative herpes simplex (HSV) type-1-derived viral vectors engineered with heterologous cytokine genes. Compared to controls, mice affected by experimental autoimmune encephalomyelitis (EAE) and i.c. injected with an HSV-1-derived vector containing the gene of the anti-inflammatory cytokine IL-4 showed a significant amelioration of clinical and pathological EAE signs. A decreased mRNA expression of the monocyte chemoattractant protein-1 (MCP-1) by mononuclear CNS-infiltrating cells was also observed. Peripheral T cells from IL-4-treated mice were not affected both in their antigen-specific proliferative response and in the cytokine secretion pattern. Our results indicate that CNS cytokine delivery with HSV-1-derived vectors is a feasible therapeutic strategy and might represent an alternative approach for the treatment of immune-mediated demyelinating diseases. Advantages of this approach over systemic cytokine administration are the high cytokine level reached within the CNS and the absence of side-effects on the peripheral immune system. The short-lasting cytokine production in the CNS after a single vector administration (4 weeks) is the limiting factor of this novel technology which, although promising, has to be improved.

Animals↗
PubMed2000

Genetically modified CD34+ cells as cellular vehicles for gene delivery into areas of angiogenesis in a rhesus model.

To develop a cellular vehicle able to reach systemically disseminated areas of angiogenesis, we sought to exploit the natural tropism of circulating endothelial progenitor cells (EPCs). Primate CD34+ EPCs were genetically modified with high efficiency and minimal toxicity using a non-replicative herpes virus vector. These EPCs localized in a skin autograft model of angiogenesis in rhesus monkeys, and sustained the expression of a reporter gene for several weeks while circulating in the blood. In animals infused with autologous CD34+ EPCs transduced with a thymidine kinase-encoding herpes virus, skin autografts and subcutaneous Matrigel pellets impregnated with vascular growth factors underwent necrosis or accelerated regression after administration of ganciclovir. Importantly, the whole intervention was perfectly well tolerated. The accessibility, easy manipulation, lack of immunogenicity of the autologous CD34+ cell vehicles, and tropism for areas of angiogenesis render autologous CD34+ circulating endothelial progenitors as ideal candidates for exploration of their use as cellular vehicles when systemic gene delivery to those areas is required. Gene Therapy (2000) 7, 43-52.

Angiogenesis Inhibitors↗
PubMed2000

Design and application of HSV vectors for neuroprotection.

Herpes simplex virus has been extensively genetically modified for gene transfer to nerve and other tissues, to create vectors that are devoid of viral gene expression and toxicity. Recombinant vectors have been engineered to express genes which protect neurons against toxic insults resulting in cell death, including nerve growth factor (NGF) and anti-apoptotic genes (eg bcl-2). This review describes experiments using HSV vectors expressing these gene products and their potential protective role in ameliorating neurodegenerative processes in animal model systems.

Gene Transfer Techniques↗
PubMed2000

Pseudotyping of glycoprotein D-deficient herpes simplex virus type 1 with vesicular stomatitis virus glycoprotein G enables mutant virus attachment and entry.

The use of herpes simplex virus (HSV) vectors for in vivo gene therapy will require the targeting of vector infection to specific cell types in certain in vivo applications. Because HSV glycoprotein D (gD) imparts a broad host range for viral infection through recognition of ubiquitous host cell receptors, vector targeting will require the manipulation of gD to provide new cell recognition specificities in a manner designed to preserve gD's essential role in virus entry. In this study, we have determined whether an entry-incompetent HSV mutant with deletions of all Us glycoproteins, including gD, can be complemented by a foreign attachment/entry protein with a different receptor-binding specificity, the vesicular stomatitis virus glycoprotein G (VSV-G). The results showed that transiently expressed VSV-G was incorporated into gD-deficient HSV envelopes and that the resulting pseudotyped virus formed plaques on gD-expressing VD60 cells, albeit at a 50-fold-reduced level compared to that of wild-type gD. This reduction may be related to differences in the entry pathways used by VSV and HSV or to the observed lower rate of incorporation of VSV-G into virus envelopes than that of gD. The rate of VSV-G incorporation was greatly improved by using recombinant molecules in which the transmembrane domain of HSV glycoprotein B or D was substituted for that of VSV-G, but these recombinant molecules failed to promote virus entry. These results show that foreign glycoproteins can be incorporated into the HSV envelope during replication and that gD can be dispensed with on the condition that a suitable attachment/entry function is provided.

Gene Targeting↗
PubMed1999

Herpes simplex virus vector-mediated expression of Bcl-2 prevents 6-hydroxydopamine-induced degeneration of neurons in the substantia nigra in vivo.

6-Hydroxydopamine (6-OHDA) is widely used to selectively lesion dopaminergic neurons of the substantia nigra (SN) in the creation of animal models of Parkinson's disease. In vitro, the death of PC-12 cells caused by exposure to 6-OHDA occurs with characteristics consistent with an apoptotic mechanism of cell death. To test the hypothesis that apoptotic pathways are involved in the death of dopaminergic neurons of the SN caused by 6-OHDA, we created a replication-defective genomic herpes simplex virus-based vector containing the coding sequence for the antiapoptotic peptide Bcl-2 under the transcriptional control of the simian cytomegalovirus immediate early promoter. Transfection of primary cortical neurons in culture with the Bcl-2-producing vector protected those cells from naturally occurring cell death over 3 weeks. Injection of the Bcl-2-expressing vector into SN of rats 1 week before injection of 6-OHDA into the ipsilateral striatum increased the survival of neurons in the SN, detected either by retrograde labeling of those cells with fluorogold or by tyrosine hydroxylase immunocytochemistry, by 50%. These results, demonstrating that death of nigral neurons induced by 6-OHDA lesioning may be blocked by the expression of Bcl-2, are consistent with the notion that cell death in this model system is at least in part apoptotic in nature and suggest that a Bcl-2-expressing vector may have therapeutic potential in the treatment of Parkinson's disease.

Animals↗
PubMed1999

Antihyperalgesic effects of infection with a preproenkephalin-encoding herpes virus.

To test the utility of gene therapeutic approaches for the treatment of pain, a recombinant herpes simplex virus, type 1, has been engineered to contain the cDNA for an opioid peptide precursor, human preproenkephalin, under control of the human cytomegalovirus promoter. This virus and a similar recombinant containing the Escherichia coli lacZ gene were applied to the abraded skin of the dorsal hindpaw of mice. After infection, the presence of beta-galactosidase in neuronal cell bodies of the relevant spinal ganglia (lacZ-containing virus) and of human proenkephalin (preproenkephalin-encoding virus) in the central terminals of these neurons indicated appropriate gene delivery and expression. Baseline foot withdrawal responses to noxious radiant heat mediated by Adelta and C fibers were similar in animals infected with proenkephalin-encoding and beta-galactosidase-encoding viruses. Sensitization of the foot withdrawal response after application of capsaicin (C fibers) or dimethyl sulfoxide (Adelta fibers) observed in control animals was reduced or eliminated in animals infected with the proenkephalin-encoding virus for at least 7 weeks postinfection. Hence, preproenkephalin cDNA delivery selectively blocked hyperalgesia without disrupting baseline sensory neurotransmission. This blockade of sensitization was reversed by administration of the opioid antagonist naloxone, apparently acting in the spinal cord. The results demonstrate that the function of sensory neurons can be selectively altered by viral delivery of a transgene. Because hyperalgesic mechanisms may be important in establishing and maintaining neuropathic and other chronic pain states, this approach may be useful for treatment of chronic pain and hyperalgesia in humans.

Animals↗
PubMed1999

Adenoviral transfer of the viral IL-10 gene periarticularly to mouse paws suppresses development of collagen-induced arthritis in both injected and uninjected paws.

Gene therapy is a promising new approach in the treatment of rheumatoid arthritis. Gene delivery to diseased joints offers the prospect of achieving high, local concentrations of a therapeutic gene product in a sustained manner, while minimizing exposure of nontarget organs. We report that a single administration of a modified adenovirus encoding the Epstein-Barr-derived homologue of IL-10 can suppress the development of disease for extended periods of time when injected locally within the periarticular tissue surrounding the ankle joints of mice with collagen type II-induced arthritis. Furthermore, we show that injection of an adenoviral vector carrying the IL-10 gene into a single paw can suppress development of arthritis in other, noninjected paws of the same individual. The systemic protection resulting from local gene therapy occurred in the absence of detectable levels of viral IL-10 in the serum. Circulating Ab levels to heterologous collagen were unaffected; however, treatment with viral IL-10 significantly suppressed the development of Abs to autologous mouse type II collagen. Thus, the treatment of a single joint by local delivery of the vIL-10 gene may protect multiple joints of the same individual while avoiding deleterious side effects often associated with systemic therapy.

Adenoviridae↗
PubMed1999

Replication-defective herpes simplex virus vectors for neurotrophic factor gene transfer in vitro and in vivo.

We report here the construction and the use of two replication-defective herpes simplex virus vectors, SH FGF-2 and TH FGF-2, which efficiently transfer and express the cDNA for fibroblast-growth-factor-2 (FGF-2) in vitro and in vivo. One mutant was deleted in the immediate-early gene encoding ICP4; the other was deleted in ICP4, ICP22 and ICP27. FGF-2--or the control gene lacZ--were inserted in tk, under control of the human cytomegalovirus immediate-early promoter. Infection of Vero cells with SH FGF-2 induced a dramatic increase in FGF-2 protein levels in the first 2 days after infection, with a rapid return to baseline levels within day 4. In contrast, infection of Vero cells with TH FGF-2 displayed FGF-2 levels progressively increasing up to days 4-5, and slowly returning to baseline. Protein extracts of cells infected with TH FGF-2 induced neuronal differentiation of PC12 cells, indicating that the newly synthesized FGF-2 was biologically active. Robust transient transgene expression was also observed in the rat hippocampus after stereotaxical inoculation of TH FGF-2, but not of TH lacZ or of SH vectors. Enhanced gene expression both in vitro and in vivo by the triple immediate-early gene deletion mutant might be attributed to reduced vector cytotoxicity. The present data suggest that TH FGF-2 is suitable for studies of FGF-2 involvement in neurological disorders.

Animals↗
PubMed1999

Herpes simplex virus type 1 vector-mediated expression of nerve growth factor protects dorsal root ganglion neurons from peroxide toxicity.

Nerve growth factor beta subunit (beta-NGF) transgene delivery and expression by herpes simplex virus type 1 (HSV-1) vectors was examined in a cell culture model of neuroprotection from hydrogen peroxide toxicity. Replication-competent (tk- K mutant background) and replication-defective (ICP4(-);tk- S mutant background) vectors were engineered to contain the murine beta-NGF cDNA under transcriptional control of either the human cytomegalovirus immediate-early gene promoter (HCMV IEp) (e.g., KHN and SHN) or the latency-active promoter 2 (LAP2) (e.g., KLN and SLN) within the viral thymidine kinase (tk) locus. Infection of rat B103 and mouse N2A neuronal cell lines, 9L rat glioma cells, and Vero cells with the KHN or SHN vectors resulted in the production of beta-NGF-specific transcripts and beta-NGF protein reaching a maximum at 3 days postinfection (p.i.). NGF protein was released into the culture media in amounts ranging from 10.83 to 352.86 ng/ml, with the highest levels being achieved in B103 cells, and was capable of inducing neurite sprouting of PC-12 cells. The same vectors produced high levels of NGF in primary dorsal root ganglion (DRG) cultures at 3 days. In contrast to HCMV IEp-mediated expression, the LAP2-NGF vectors showed robust expression in primary DRG neurons at 14 days. The neuroprotective effect of vector produced NGF was assessed by its ability to inhibit hydrogen peroxide-induced neuron toxicity in primary DRG cultures. Consistent with the kinetics of vector-mediated NGF expression, HCMV-NGF vectors were effective in abrogating the toxic effects of peroxide at 3 but not 14 days p.i. whereas LAP2-NGF vector transduction inhibited apoptosis in DRG neurons at 14 days p.i. but was ineffective at 3 days p.i. Similar kinetics of NGF expression were observed with the KHN and KLN vectors in latently infected mouse trigeminal ganglia, where high levels of beta-NGF protein expression were detected at 4 wks p.i. only from the LAP2; HCMV-NGF-driven expression peaked at 3 days but could not be detected during HSV latency at 4 weeks. Together, these results indicate that (i) NGF vector-infected cells produce and secrete mature, biologically active beta-NGF; (ii) vector-synthesized NGF was capable of blocking peroxide-induced apoptosis in primary DRG cultures; and (iii) the HCMV-IEp functioned to produce high levels of NGF for several days; but (iv) only the native LAP2 was capable of long-term expression of a therapeutic gene product in latently infected neurons in vivo.

Animals↗
PubMed1999

Genetic studies exposing the splicing events involved in herpes simplex virus type 1 latency-associated transcript production during lytic and latent infection.

Herpes simplex virus type 1 (HSV-1) establishes latency in sensory neurons, a state in which the viral lytic genes are silenced and only the latency locus is transcriptionally active, producing the 2. 0- and 1.5-kb latency-associated transcripts (LATs). Previous experimental evidence indicates that the LATs are stable introns, and it has been reported that LAT formation is abolished by debilitating substitution mutations in the predicted splice sites during lytic infection but not latency (J. L. Arthur et al., J. Gen. Virol. 79:107-116, 1998). We have independently studied a set of deletion mutations to explore the roles of the proposed splice sites during lytic and latent infection. HSV-1 mutant viruses missing the invariant intron-terminal 5'-G(T/C) or 3'-AG dinucleotides were analyzed for LAT formation during lytic infection in vitro, when only the 2-kb LAT is produced, and during latency in mouse trigeminal ganglia, where both LATs are expressed. Northern blot analysis of total RNAs from different productively infected cell lines showed that the lytic (2-kb) LAT was not expressed by the various splice site deletion mutants. In vivo studies using a mouse eye model of latency similarly showed that the latent (2- and 1. 5-kb) LATs were not expressed by the mutants. PCR analysis with primers flanking the LAT sequence revealed the expected splice junction for LAT excision in RNA from sensory neurons latently infected with wild-type but not mutant virus. Using a virus mutant deleted in the splicing signals flanking the 556-bp region of LAT whose absence distinguishes the 1.5- and 2-kb LATs, we observed selective elimination of 1.5-kb LAT expression in latency, supporting previous suggestions that the internal region is removed by splicing. Taken together, these results demonstrate that the 2-kb LAT is formed during both lytic and latent infection by splicing at the predicted splice sites and that an additional splicing event is involved in the latency-restricted production of the 1.5-kb LAT. We have also mapped the 3' end of the lytic 2-kb LAT and discuss our results in the context of previous models addressing the unusual stability of the LATs.

Animals↗
PubMed1998

Enhanced tumor cell killing in the presence of ganciclovir by herpes simplex virus type 1 vector-directed coexpression of human tumor necrosis factor-alpha and herpes simplex virus thymidine kinase.

Past studies have documented the promise of herpes simplex virus type 1 (HSV-1) thymidine kinase (TK) suicide gene therapy as a potential antitumor treatment. HSV-TK converts the pro-drug ganciclovir (GCV) into a toxic nucleotide analogue, the incorporation of which into cellular DNA blocks cell proliferation. In this report, we have examined the hypothesis that the effectiveness of HSV-TK suicide gene therapy can be enhanced by coexpression of the antitumor cytokine human tumor necrosis factor-alpha (TNF-alpha) from the same replication-defective HSV-1 vector. In vitro testing demonstrated that TNF-alpha expression from this vector potentiated the killing of both TNF-alpha-sensitive L929 tumor cells and TNF-alpha-resistant U-87 MG cells in the presence of GCV. Furthermore, treatment of established intradermal L929 tumors in vivo with the TNF-alpha/TK vector and GCV resulted in prolonged animal survival compared with treatment with parental HSV-TK vector in the presence or absence of GCV. Treatment of intracerebral U-87 MG tumors showed a clear benefit of TK therapy, but a significant further increase in survival using the TNF-alpha vector could not be demonstrated. We found that potentiation of cell killing in vitro required intracellular TNF-alpha because purified protein added to the culture medium of cells infected with HSV-TK vector failed to have the same effect. Accordingly, potentiation in vivo should depend on efficient infection, but immunohistochemical analysis indicated that virus administration by U-87 MG intratumoral injection was inadequate, resulting in an estimated <1% infection of all tumor cells. Moreover, the majority of infected tumor cells were localized at the tumor margin. Together, these results suggest that TNF-enhanced tk gene therapy should provide a useful treatment for TNF-alpha-sensitive tumors and perhaps also for TNT-alpha-resistant tumors if vector delivery can be improved to increase the percentage of transduced tumor cells.

Animals↗
PubMed1998

Efficient gene delivery into epstein-barr virus (EBV)-transformed human B cells mediated by replication-defective herpes simplex virus-1 (HSV-1): A gene therapy model for EBV-related B cell malignancy.

Subgroups of the B cell malignancies are known to be associated with Epstein-Barr virus (EBV) infection, especially in immunocompromised patients. These are fatal and refractory to conventional antineoplastic therapy. B cells are usually post-mitotic cells and even mitogen activated or transformed B cells have shown relative resistance against viral mediated gene transfer. To address this issue, we employed a replication-defective herpes simplex virus-1 (HSV-1) to mediate gene transfer into EBV-transformed B cells. The virus expresses the herpes simplex virus thymidine kinase (HSV-TK) and the E. coli lacZ reporter genes and is designated T0Z.1. We used the lymphoblastoid cell line SWEIG as a model for human EBV-related B cell malignancy. This cell line was established by in vitro EBV infection of primary human peripheral blood mononuclear cells. When SWEIG cells were infected with T0Z.1, X-gal staining revealed lacZ expression in more than 20% cells even at multiplicity of infection (MOI) as low as 1 and the expression persisted for at least one week. Ganciclovir (GCV) administration after T0Z.1 infection effectively decreased the number of the infected tumor cells in a dose-responsive manner. Viral toxicity was analyzed by cell proliferation assay (MTS assay) and found to be little even at 10 MOI infection. Three MOI of the virus yielded maximum antineoplastic effect and more than 50% tumor cells were killed by HSV-TK/GCV. These results suggest the potential utility of replication-defective HSV-1 for the treatment of EBV-related B cell malignancies.

Antiviral Agents↗
PubMed1998

Herpes virus-mediated preproenkephalin gene transfer to the amygdala is antinociceptive.

To evaluate the role of the amygdala in pain modulation and opioid-mediated antinociception, a recombinant, replication-defective herpes virus carrying the human preproenkephalin cDNA was injected bilaterally into the rat amygdala. Four days after gene delivery nociceptive behavior was assessed by the formalin test. Rats infected with the virus expressing preproenkephalin showed a selective, naloxone-reversible abolition of phase 2 flinching behavior compared to rats infected with a control virus. The results implicate the amygdala in the control of pain and in opioid analgesia and demonstrate the use of recombinant herpes viruses as tools for studying gene function in specific neural pathways of the central nervous system.

Amygdala↗
PubMed1998

Modulation of mucosal and systemic immunity by enteric administration of nonreplicating herpes simplex virus expressing cytokines.

In this report the ability of enteric immunization with recombinant replication deficient (ICP4-/-) HSV expressing IFN gamma to generate protection and modulate mucosal and systemic immunity was evaluated. ICP4-/-HSV, ICP4-/-HSV expressing IL4, live replicating, and uv HSV were used as controls. Following enteric administration of live HSV, a Th1 cytokine response was induced in the spleen, while both Th1 and notable Th2 cytokine production were detected at mucosal sites. Modulation of mucosal and systemic immune response was achieved when nonreplicating recombinant HSV viruses expressing cytokines were used. Compared to the control replication defective viruses, decreased frequency of Th2 cytokine producing cells in Peyer's patches was observed following enteric administration of nonreplicating HSV expressing IFN gamma. When IFN gamma expressing virus was given enterically, modulation was observed at the systemic level, measured by ELISPOT for cytokine producing cells, ELISA from the in vitro restimulated splenic cell cultures, and by the increase of the IgG2a/IgG1 ratio in the serum. This report provides evidence that replication defective viruses expressing cytokine genes in contrast to uv HSV, are immunogenic when administered enterically and can generate significant immunomodulatory effects at the mucosal and systemic levels.

Animals↗
PubMed1998

High-efficacy thymidine kinase gene transfer to ovarian cancer cell lines mediated by herpes simplex virus type 1 vector.

In this report, a replication-defective herpes simplex virus type 1 (HSV-1) vector has been employed to deliver the Escherichia coli LacZ and HSV thymidine kinase (HSVtk) genes to six human ovarian carcinoma cell lines and the efficacy of gene transfer compared to that of adenoviral vectors in vitro. The transduction efficiency of the LacZ-containing virus TOZ.1 was evaluated qualitatively and quantitatively following infection of the different ovarian cancer cell lines. The therapeutic ability of the HSV-T3 vector, which contains the HSVtk gene, was additionally investigated in comparison to the AdCMVHSVTK. Our results show that HSV-1-mediated gene transfer is quantitatively superior to adenoviral vector in five of the six ovarian cancer cell lines at a 100-fold lower dose in vitro. Our preliminary studies suggest that HSV-1 may be a promising alternative vector for ovarian cancer gene therapy.

Cell Division↗
PubMed1998

Implications of maturation for viral gene delivery to skeletal muscle.

Different viral vectors have been analyzed as gene delivery vehicles to skeletal muscle for potentially therapeutic purposes. In this review, we evaluate the application of retroviral, adenoviral, and herpes simplex viral vectors to deliver genes to skeletal muscle and focus on the dramatic loss of viral transduction detected throughout muscle maturation. Recent results suggested that there are several factors involved in the reduced viral transducibility of mature skeletal muscle: muscle cells become post-mitotic in an early stage, the extracellular matrix develops into a physical barrier, and a loss of myoblast mediation occurs since myoblasts progressively become quiescent. Approaches to improve viral gene delivery to mature skeletal muscle may include the use of particular enzymes to increase the permeability of the extracellular matrix, the pre-treatment of the muscle with a myonecrotic agent to induce myoblast mediation, or the application of the myoblast-mediated ex vivo gene transfer.

Adenoviridae↗
PubMed1998

Drug inducible transgene expression in brain using a herpes simplex virus vector.

The ability to regulate transgene expression is likely to be important in the use of gene transfer to treat diseases of the central nervous system (CNS). In order to achieve regulatable gene expression we created a replication-incompetent genomic herpes simplex vector containing a RU486-inducible transactivator and a lacZ reporter gene under transcriptional control of a minimal promoter. Reporter gene expression from the vector was regulated by administration of RU486 in vitro and in vivo. In cell culture half maximal expression was achieved with 10(-8) M RU486, and maximal expression was achieved by 24 h. Following stereotactic inoculation of the vector into rat hippocampus, expression was increased 150-fold by i.p. administration of RU486. This demonstrates that the RU486 system functions as a tight on/off switch for regulating expression of a transgene delivered to the brain via an HSV vector.

Animals↗