Biomedical subjects

G Y Gillespie

Publications and source records attributed to G Y Gillespie.

At least 19 recordsLinked to original sources

PubMed2006

Directing adenovirus across the blood-brain barrier via melanotransferrin (P97) transcytosis pathway in an in vitro model.

Adenovirus serotype 5 (Ad5) is widely used in the development of gene therapy protocols. However, current gene therapy strategies involving brain are mostly based on intra-cranial injection. A major obstacle for systemically administered vectors to infect brain tissue is the blood-brain barrier (BBB). One strategy to cross the BBB is transcytosis, a transcellular transport process that shuttles a molecule from one side of the cell to the other side. Recently, melanotransferrin (MTf)/P97 was found to be able to cross the BBB and accumulate in brain. We thus hypothesize that re-directing Ad5 vectors to the MTf transcytosis pathway may facilitate Ad5 vectors to cross the BBB. To test this hypothesis, we constructed a bi-specific adaptor protein containing the extracellular domain of the coxsackie-adenovirus receptor (CAR) and the full-length melanotransferrin (sCAR-MTf), and investigated its ability to re-direct Ad5 vectors to the MTf transcytosis pathway. We found this adaptor protein could re-direct Ad5 to the MTf transcytosis pathway in an in vitro BBB model, and the transcytosed Ad5 viral particles retained their native infectivity. The sCAR-MTf-mediated Ad5 transcytosis was temperature- and dose dependent. In addition, we examined the directionality of sCAR-MTf-mediated Ad5 transcytosis, and found the efficiency of apical-to-basal transcytosis was much higher than that of basal-to-apical direction, supporting a role of this strategy in transporting Ad5 vectors towards the brain. Taken together, our study demonstrated that re-directing Ad5 to the MTf transcytosis pathway could facilitate gene delivery across the BBB.

Adenoviridae↗

PubMed2006

Engineered herpes simplex virus expressing bacterial cytosine deaminase for experimental therapy of brain tumors.

Lack of effective therapy of primary brain tumors has promoted the development of novel experimental approaches utilizing oncolytic viruses combined with gene therapy. Towards this end, we have assessed a conditionally replication-competent, gamma(1)34.5-deleted herpes simplex virus type 1 (HSV-1) expressing cytosine deaminase (CD) for treatment of malignant brain tumors. Our results are summarized as follows: (i) a recombinant HSV (M012) was constructed in which both copies of the gamma(1)34.5 gene were replaced with the bacterial CD gene, under the control of the cellular promoter Egr-1; (ii) M012-infected cells in vitro efficiently convert 5-fluorocytosine (5-FC) to 5-fluorouracil, thereby enhancing cytotoxicity of neighboring, uninfected cells; (iii) both direct and bystander cytotoxicity of murine neuroblastoma and human glioma cell lines after infection with M012 were demonstrated; (iv) direct intracerebral inoculation of A/J mice demonstrated lack of neurotoxicity at doses similar to G207, a gamma(1)34.5-deleted HSV with demonstrated safety in human patient trials and (v) intratumoral injection of M012 into Neuro-2a flank tumors in combination with 5-FC administration significantly reduced tumor growth versus tumors treated with R3659 combined with 5-FC, or treated with M012 alone. Thus, M012 is a promising new oncolytic HSV vector with an enhanced prodrug-mediated, antineoplastic effect that is safe for intracranial administration.

Animals↗

PubMed2002

The use of a genetically engineered herpes simplex virus (R7020) with ionizing radiation for experimental hepatoma.

The herpes simplex virus (HSV) recombinant virus R7020 is an attenuated virus designed as a candidate for immunization against both HSV-1 and HSV-2 infections. It was extensively tested in an experimental animal system and in a healthy human adult population without significant untoward effects. We report on the use of R7020 with ionizing radiation as an oncolytic agent for hepatomas. Two hepatoma cell lines were studied, Hep3B and Huh7. R7020 replicated to higher titers in Hep3B cells than in Huh7 cells. Tissue culture studies correlated with hepatoma xenograft responses to R7020. R7020 was more effective in mediating Hep3B tumor xenograft regression compared with Huh7. Ionizing radiation combined with R7020 also showed differential results in antitumor efficacy between the two cell lines in tumor xenografts. Ionizing radiation enhanced the replication of R7020 in Hep3B xenografts. Moreover, the combination of ionizing radiation and virus caused a greater regression of xenograft volume than either R7020 or radiation alone. Ionizing radiation had no effect on the replication of R7020 virus in Huh7 xenografts. These results indicate that a regimen involving infection with an appropriate herpesvirus such as R7020 in combination with ionizing radiation can be highly effective in eradicating certain tumor xenografts.

Animals↗

PubMed2001

RNA replicons derived from poliovirus are directly oncolytic for human tumor cells of diverse origins.

The failure and/or toxicity of conventional therapies for many types of human cancers underscore the need for development of safe and effective alternative treatments. Toward this goal, we describe the direct oncolytic activity of RNA-based vectors derived from poliovirus, termed replicons, which are genetically incapable of producing infectious virus. These replicons are cytopathic in vitro for human tumor cells originating from brain, breast, lung, ovary, and skin (melanoma). The cytopathic effects in a malignant glioma cell line were associated with nuclear DNA condensation, indicative of cells undergoing apoptosis. Injection of replicons into established xenograft flank tumors in scid mice resulted in oncolytic activity and extended survival. Inoculation of replicons into established intracranial xenograft tumors in scid mice resulted in tumor infection within 8 h and extended survival. Histological analysis revealed that replicons had infected tumor cells at the site of inoculation and, most importantly, diffused to infect tumor cells that had metastasized from the initial site of implantation. The wide spectrum of cytopathic activity for human tumors combined with effective distribution after in vivo inoculation establishes the therapeutic potential of poliovirus replicons for a variety of cancers.

Animals↗

PubMed2001

Evidence for peroxynitrite-mediated modifications to p53 in human gliomas: possible functional consequences.

Based on previous findings of increased nitric oxide synthase (NOS) expression in human gliomas (4), we hypothesized that peroxynitrite, a highly reactive metabolite of nitric oxide (NO) and superoxide (O(*-)(2)), might be increased in these tumors in vivo. Here we demonstrate that nitrotyrosine (a footprint of peroxynitrite protein modification) is present in human malignant gliomas. Furthermore, we show that p53, a key tumor suppressor protein, has evidence of peroxynitrite-mediated modifications in gliomas in vivo. Experiments in vitro demonstrate that peroxynitrite treatment of recombinant wild-type p53 at physiological concentrations results in formation of higher molecular weight aggregates, tyrosine nitration, and loss of specific DNA binding. Peroxynitrite treatment of human glioma cell lysates similarly resulted in selective tyrosine nitration of p53 and was also associated with loss of p53 DNA binding ability. These data indicate that tyrosine nitration of proteins occurs in human gliomas in vivo, that p53 may be a target of peroxynitrite in these tumors, and that physiological concentrations of peroxynitrite can result in a loss of p53 DNA binding ability in vitro. These findings raise the possibility that peroxynitrite may contribute to loss of wild-type p53 functional activity in gliomas by posttranslational protein modifications.

Blotting, Western↗

PubMed2001

pH alterations "reset" Ca2+ sensitivity of brain Na+ channel 2, a degenerin/epithelial Na+ ion channel, in planar lipid bilayers.

Members of the degenerin/epithelial Na(+) channel superfamily of ion channels subserve many functions, ranging from whole body sodium handling to mechanoelectrical transduction. We studied brain Na(+) channel 2 (BNaC-2) in planar lipid bilayers to examine its single channel properties and regulation by Ca(2+). Upon incorporation of vesicles made from membranes of oocytes expressing either wild-type (WT) BNaC-2 or BNaC-2 with a gain-of-function (GF) point mutation (G433F), functional channels with different properties were obtained. WT BNaC-2 resided in a closed state with short openings, whereas GF BNaC-2 was constitutively activated; a decrease in the pH in the trans compartment of the bilayer activated WT BNaC-2 and decreased its permeability for Na(+) over K(+). Moreover, these maneuvers made the WT channel more resistant to amiloride. In contrast, GF BNaC-2 did not respond to a decrease in pH, and its amiloride sensitivity and selectivity for Na(+) over K(+) were unaffected by this pH change. Buffering the bathing solutions with EGTA to reduce the free [Ca(2+)] to <10 nm increased WT single channel open probability 10-fold, but not that of GF BNaC-2. Ca(2+) blocked both WT and GF BNaC-2 in a dose- and voltage-dependent fashion; single channel conductances were unchanged. A drop in pH reduced the ability of Ca(2+) to inhibit these channels. These results show that BNaC-2 is an amiloride-sensitive sodium channel and suggest that pH activation of these channels could be, in part, a consequence of H(+) "interference" with channel regulation by Ca(2+).

Acid Sensing Ion Channels↗

PubMed2001

Fas-induced expression of chemokines in human glioma cells: involvement of extracellular signal-regulated kinase 1/2 and p38 mitogen-activated protein kinase.

Fas transduces not only apoptotic signals through various pathways but also angiogenic and proinflammatory responses in vivo. Human glioma cells express Fas although sensitivity to Fas-mediated cell death is variable, suggesting that Fas may have functions other than apoptosis in these cells. In this study, we addressed alternative functions of Fas expressed on human gliomas by Fas ligation in three human glioma cell lines, CRT-MG, U373-MG, and U87-MG, and the in vivo expression of Fas and chemokines in human glioblastoma multiforme (GBM). Herein, we demonstrate that: (a) stimulation with agonistic anti-Fas monoclonal antibody CH-11 and human recombinant soluble Fas ligand induces expression of the CC chemokine MCP-1 and the CXC chemokine interleukin-8 by human glioma cell lines at the mRNA and protein levels in a dose- and time-dependent manner; (b) selective pharmacological inhibitors of MEK1 (U0126 and PD98059) and p38 mitogen-activated protein kinase (MAPK) (SB202190) suppress Fas-mediated chemokine expression in a dose-dependent manner; (c) Fas ligation on human glioma cells leads to activation of both extracellular signal-regulated kinases ERK1/ERK2 and p38 MAPK; and (d) GBM samples express higher levels of Fas compared with normal control brain, which correlates with increased interleukin 8 expression. These findings indicate that Fas ligation on human glioma cells leads to the selective induction of chemokine expression, which involves the ERK1/ERK2 and p38 MAPK signaling pathways. Therefore, the Fas-Fas ligand system in human brain tumors may be involved not only in apoptotic processes but also in the provocation of angiogenic and proinflammatory responses.

Adult↗

PubMed2001

HuR, a RNA stability factor, is expressed in malignant brain tumors and binds to adenine- and uridine-rich elements within the 3' untranslated regions of cytokine and angiogenic factor mRNAs.

Tumors of the central nervous system (CNS) often have sustained expression of labile genes, including angiogenic growth factors and immunosuppressive cytokines, which promote tumor progression. Stabilization of the RNA transcripts for these genes, such as vascular endothelial growth factor (VEGF), is an important molecular pathway for this up-regulation. HuR, a member of the Elav family of RNA-binding proteins, has been implicated in this pathway through its binding to adenine and uridine (AU)-rich stability elements (ARE) located in the 3' untranslated regions (3'-UTRs) of the mRNA. Whereas three of the Elav family members (Hel-N1, HuC, and HuD) are restricted to young and mature neurons, HuR is more broadly expressed, including proliferating cells of the developing CNS. Because RNA stabilization of labile genes may promote tumor growth, we analyzed and compared the expression pattern of HuR in 35 freshly resected and cultured CNS tumors to determine whether there was any correlation with tumor grade or histological type. We found that HuR mRNA was consistently expressed in all of the tumors, regardless of cell origin or degree of malignancy. Using a novel HuR-specific polyclonal antibody, we found that strong HuR protein expression was limited to high-grade malignancies (glioblastoma multiforme and medulloblastoma). Within the glioblastoma multiforme, prominent HuR expression was also detected in perinecrotic areas in which angiogenic growth factors are up-regulated. To further define its role as a potential RNA stabilizer, we analyzed whether HuR could bind to the stability motifs within the 3'-UTRs of cytokines and growth factors linked to brain tumor progression. We used a novel ELISA-based RNA binding assay and focused on the 3'-UTRs of angiogenic factors VEGF, COX-2, and (interleukin) IL-8 as well as the immunomodulating factors IL-6, transforming growth factor (TGF)-beta and tumor necrosis factor (TNF)-alpha as potential RNA ligands. Our results indicated overall a very high binding affinity to these RNA targets. A comparison of these ligands revealed a hierarchy of binding affinities with the angiogenic factors, and TGF-beta showing the highest (Kd of 1.8-3.4 nM), and TNF-alpha the lowest (Kd of 18.3 nM). The expression pattern of HuR, coupled with the RNA binding data, strongly suggests a role for this protein in the posttranscriptional regulation of these genes in CNS tumors.

3' Untranslated Regions↗

PubMed2001

Differential gene expression profiling in human brain tumors.

Gene expression profiling of three human temporal lobe brain tissue samples (normal) and four primary glioblastoma multiforme (GBM) tumors using oligonucleotide microarrays was done. Moreover, confirmation of altered expression was performed by whole cell patch clamp, immunohistochemical staining, and RT-PCR. Our results identified several ion and solute transport-related genes, such as N-methyl-d-aspartate (NMDA) receptors, alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA)-2 receptors, GABA(A) receptor subunits alpha3, beta1, beta2, and beta3, the glutamate transporter, the glutamate/aspartate transporter II, the potassium channel K(V)2.1, hK(V)beta3, and the sodium/proton exchanger 1 (NHE-1), that are all downregulated in the tumors compared with the normal tissues. In contrast, aquaporin-1, possibly aquaporins-3 and -5, and GLUT-3 message appeared upregulated in the tumors. Our results also confirmed previous work showing that osteopontin, nicotinamide N-methyltransferase, murine double minute 2 (MDM2), and epithelin (granulin) are upregulated in GBMs. We also demonstrate for the first time that the cytokine and p53 binding protein, macrophage migration inhibitory factor (MIF), appears upregulated in GBMs. These results indicate that the modulation of ion and solute transport genes and heretofore unsuspected cytokines (i.e., MIF) may have profound implications for brain tumor cell biology and thus may identify potential useful therapeutic targets in GBMs.

Aquaporin 1↗

PubMed2001

VEGF-D is an X-linked/AP-1 regulated putative onco-angiogen in human glioblastoma multiforme.

BACKGROUND: Glioblastoma multiforme (GBM) is a hypervascularized and locally infiltrating brain tumor of astroglial origin with a very poor prognosis. An X-linked c-fos oncogene-inducible mitogenic, morphogenic, and angiogenic factor, endothelial growth factor-D (VEGF-D), is the newest mammalian member of VEGF family. We analyzed VEGF-D in GBM because of its high angiogenic potential and its linkage to the X chromosome. MATERIALS AND METHODS: Nonmalignant brain and GBM tissue sections as well as GBM cell lines were analyzed by immunofluorescence for the expression of VEGF-D, factor VIII (endothelial cell marker), glial-fibrillary acidic protein (GFAP) (astrocytic cell lineage cytoplasmic marker), and several Fos family transcription factors, including c-Fos and Fra-1. The proteins were also detected by Western blots. The differences between genotypes of normal brain and GBM cells were examined by cDNA microarrays. RESULTS AND CONCLUSIONS: GBM expressed ubiquitously VEGF-D, which colocalized with GFAP. Contrary to our expectations, low levels of c-Fos were detected in GBM cells. However, we identified another Fos family member, Fra-1, together with its transcriptional activation partner, c-Jun, as being stably up-regulated in GBM cells. Furthermore, we demonstrated that a fra-1 transgene induced VEGF-D expression in cultured cells and GBM cell stimulation evoked a sustained increase in both Fra-1 and VEGF-D levels. This study reveals that an up-regulation of AP-1 factors may be a hallmark of GBM. Because VEGF-D activates VEGF receptor 2 and 3, receptors important for tumor angiogenesis, it may represent an X-linked/AP-1-regulated onco-angiogen in human GBM. The VEGF-D system and AP-1 activity appear to be very attractive targets for new molecular diagnostics and rational molecular anti-cancer therapies.

Animals↗

PubMed2001

Genetically engineered human herpes simplex virus in the treatment of brain tumours.

Central nervous system malignancies--particularly glioblastoma multiforme--pose significant problems for the development of novel therapeutics. In the absence of advances with standard surgical and chemotherapeutic approaches, the utilization of genetically engineered viruses--both as direct oncolytic agents (virus therapy) and for the delivery of foreign proteins (gene therapy)--represents a significant advance in the experimental approach to the management of patients with incurable tumours. Among other viruses, herpes simplex virus (HSV) offers an opportunity to influence the replication of tumour cells directly within the central nervous system. The propensity for HSV to replicate in tumour cells, and its large coding capacity, provide an experimental model for the development of novel therapeutics. The status of these experimental approaches and Phase I studies are summarized.

Animals↗

PubMed2000

Engineered herpes simplex virus expressing IL-12 in the treatment of experimental murine brain tumors.

Genetically engineered, neuroattenuated herpes simplex viruses (HSVs) expressing various cytokines can improve survival when used in the treatment of experimental brain tumors. These attenuated viruses have both copies of gamma(1)34.5 deleted. Recently, we demonstrated increased survival of C57BL/6 mice bearing syngeneic GL-261 gliomas when treated with an engineered HSV expressing IL-4, as compared with treatment with the parent construct (gamma(1)34. 5(-)) alone or with a virus expressing IL-10. Herein, we report construction of a conditionally replication-competent mutant expressing both subunits of mIL-12 (M002) and its evaluation in a syngeneic neuroblastoma murine model. IL-12 induces a helper T cell subset type 1 response, which may induce more durable antitumor effects. In vitro studies showed that, when infected with M002, both Vero cells and murine Neuro-2a neuroblastoma cells produced physiologically relevant levels of IL-12 heterodimers, as determined by ELISA. M002 was cytotoxic for Neuro-2a cells and human glioma cell lines U251MG and D54MG. Neurotoxicity studies, as defined by plaque-forming units/LD(50), performed in HSV-1-sensitive A/J strain mice found that M002 was not toxic even at high doses. When evaluated in an intracranial syngeneic neuroblastoma murine model, median survival of M002-treated animals was significantly longer than the median survival of animals treated with R3659, the parent gamma(1)34.5(-) mutant lacking any cytokine gene insert. Immunohistochemical analysis of M002-treated tumors identified a pronounced influx of CD4(+) T cells and macrophages as well as CD8(+) cells when compared with an analysis of R3659-treated tumors. We conclude that M002 produced a survival benefit via oncolytic effects combined with immunologic effects meditated by helper T cells of subset type 1.

Animals↗

PubMed2000

Human malignant glioma therapy using anti-alpha(v)beta3 integrin agents.

Glioblastoma multiforme (GBM) is the most frequent malignant brain tumor in adults and is invariably fatal. We have investigated the effect of cyclo-(Arg-Gly-Asp-D-Phe-Val) (cRGDfV) peptide on survival of human malignant glioma cells in vitro and in vivo. Immunofluorescent analyses revealed the presence of alpha(v)beta3 integrin on U-87MG and U-373MG cells, but minimal expression on U-251MG cells. Treatment of U-87MG and U-373MG cells in vitro with cRGDfV (20 microg/ml), but not the linear peptide, resulted in the appearance of rounded and loosely attached cells with subsequent cell death. By comparison, neither this cyclic peptide nor its linear homolog had any significant effect on growth and morphology of U-251MG cells. The death of cRGDfV-treated (20 microg/ml) glioma cells was blocked by pretreatment (10 microM) of cells with DEVD-FMK and LEHD-FMK, inhibitors of caspase-3 and caspase-9, respectively. Moreover, when glioma cells grown as spheroids were treated with cRGDfV (50 microg/ml), spheroid formation was markedly reduced. Further, treatment of intracranial U-87MG tumors in scid mice with cyclic peptide significantly (p < 0.001) prolonged their survival. These results indicated (i) that cRGDfV induced apoptosis of human glioma cells by binding alpha(v)beta3 integrin expressed on their cell surfaces and (ii) that cRGDfV may be an effective and non-toxic direct anti-tumor therapy for alpha(v)beta3-expressing GBMs.

Animals↗

PubMed2000

Expression of a restrictive receptor for interleukin 13 is associated with glial transformation.

We have previously documented that the vast majority of high-grade gliomas over-express binding sites for interleukin 13 (IL13) in situ. We now extend this analysis to evaluate the distribution of the binding of IL13 among other brain tumors. Tumor specimens from patients with low-grade gliomas, oligodendrogliomas, ependymomas, pilocytic astrocytomas, gliosarcomas, medulloblastomas, meningiomas, and metastases to the brain were analyzed and compared to a new series of glioblastoma multiforme (GBM) samples. Serial tumor tissue sections were incubated with 125I-labeled (i) IL13, (ii) antibody against transferrin (Tf) receptor, and (iii) epidermal growth factor (EGF). Most (17/18) GBMs stained specifically for IL13 binding sites while sections from 3/11 low-grade gliomas, 5/5 high-grade gliomas (grade III), 3/5 oligodendrogliomas (all three were anaplastic), and 1/2 gliosarcomas also showed specific binding for IL13. We did not detect IL13 binding sites in medulloblastomas (0/4) and found them only in 2/20 meningiomas. Metastases to the brain (4/12, i.e., lung adenocarcinomas and renal cell carcinoma) showed some binding of 125I-IL13. The presence of receptors for Tf was ubiquitous among all studied tumors while EGF receptor expression was much more variable. Since it appears that primarily the least differentiated forms of gliomas possess IL13 binding sites in abundance, it is plausible that IL 13 receptor expressed in low-grade gliomas might be a prognostically significant marker associated with their progression to high-grade gliomas. Finally, we demonstrate that the glioma-associated IL13 receptor is truly more restrictive in nature also due to its selective representation among brain tumors of glial origin.

Adenocarcinoma↗

PubMed2000

Immunotherapy of a murine T cell lymphoma localized to the brain.

Mouse YC8 T cell lymphoma was used as a model to determine whether an effective immunotherapy procedure could be devised for the treatment of lymphoma localized to the brain. Implantation of 5 x 10(4) YC8 cells into the left cerebral hemisphere induced rapid loss of the animal's body weight. Severe loss of weight and early deaths were observed in the untreated control group. Although resistance can be conferred to the brain by immunization of naive BALB/c mice, adoptive chemoimmunotherapy procedures were surprisingly not effective in inducing remissions in animals with lymphoma confined to the brain. Even passive transfer of effector cells from immunized, tumor resistant donor animals combined with systemic IL-2 treatment did not impart resistance to recipients with brain tumors. However, regression of the intracranial tumor and apparent cures could be accomplished, when ex vivo cultured effector cells were transferred intravenously.

Adoptive Transfer↗

PubMed2000

Conditionally replicating herpes simplex virus mutant, G207 for the treatment of malignant glioma: results of a phase I trial.

G207 is a conditionally replicating derivative of herpes simplex virus (HSV) type-1 strain F engineered with deletions of both gamma(1)34.5 loci and a lacZ insertion disabling the UL39 gene. We have demonstrated the efficacy of G207 in treating malignant glial tumors in athymic mice, as well as the safety of intracerebral G207 inoculation in mice and in Aotus nancymai. We sought to determine the safety of G207 inoculation into cerebral malignant glial tumors in humans. Criteria for inclusion into this dose-escalation study were the diagnosis of histologically proven malignant glioma, Karnofsky score > or = 70, recurrence despite surgery and radiation therapy, and an enhancing lesion greater than 1 cm in diameter. Serial magnetic resonance images were obtained for volumetric analysis. The trial commenced at a dose of 10(6) plaque forming units (p.f.u.) inoculated at a single enhancing site and was completed when the 21st patient was inoculated with 3x10(9) p.f.u. at five sites. While adverse events were noted in some patients, no toxicity or serious adverse events could unequivocally be ascribed to G207. No patient developed HSV encephalitis. We found radiographic and neuropathologic evidence suggestive of anti-tumor activity and long-term presence of viral DNA in some cases.

Adult↗

PubMed2000

Targeted foreign gene expression in spinal cord neurons using poliovirus replicons.

A hallmark of poliovirus is the propensity to infect and replicate in spinal cord neurons of the central nervous system. Previously, we characterized a poliovirus self-replicating RNA genome (replicon), which encodes firefly luciferase in place of the capsid genes. This replicon is encapsidated into an authentic poliovirion by providing the poliovirus capsid protein in trans. The amount of enzymatically active luciferase in cells infected with this replicon correlated with the infectious dose. To begin to characterize the in vivo infectious potential of replicons, we have inoculated mice transgenic for the human receptor for poliovirus (PVR), either intracranially or intraspinally, with the replicon encoding luciferase. Wild-type poliovirus delivered to PVR mice via intracranial or intraspinal routes resulted in paralysis and death. Replicon preparations were shown by a sensitive biological assay to be free of infectious poliovirus. Neither intracranial nor intraspinal inoculation of the replicon encoding luciferase resulted in any obvious paralysis or disease symptoms. Following intraspinal inoculation with replicons encoding luciferase, luciferase enzyme activity was detected at 4 h post-inoculation, with peak activity at approximately 8 h post-inoculation; by 48 - 72 h, the luciferase activity had returned to background levels. Luciferase activity was detected in spinal cord predominantly near the site of inoculation, although activity was detected anterior and posterior to the site of inoculation, indicating that replicons undergo limited movement within the CNS presumably via the cerebrospinal fluid. In stark contrast to poliovirus though, inoculation of replicons into the spinal cords of PVR mice did not result in noticeable pathogenesis. Using immunofluorescence with antibodies to double-stain for replicons and neurons, we determined that replicons exclusively infect the neurons of the spinal cord, with the expression of the luciferase and replicon proteins confined to the cytoplasm of the infected cells. Replicons, then, possess the identical capacity for infection of spinal cord neurons in vivo as poliovirus. The lack of discernible neuronal destruction following replicon inoculation into the spinal cord suggests that some of the pathogenesis observed during a poliovirus infection might not be due entirely to primary infection of neurons. Finally, the results of this study point to future use of replicons as a means to target recombinant protein expression to neurons in the spinal cord.

Animals↗

PubMed1999

Replication-competent, nonneuroinvasive genetically engineered herpes virus is highly effective in the treatment of therapy-resistant experimental human tumors.

A genetically engineered, nonneurotropic herpes simplex virus (R7020) with a proven safety profile in both animals and humans was found effective in the treatment of large xenotransplanted tumors arising from a radiation- and chemotherapy-resistant human epidermoid carcinoma and a hormone-refractory prostate adenocarcinoma. R7020 replicated to high titer and caused rapid regression of the human tumor xenografts. Tumor destruction was accelerated in animals given both R7020 and fractionated ionizing radiation. Tumors arising from cells surviving one treatment with R7020 were fully susceptible to a second dose of virus. We conclude R7020 is an effective antitumor agent for non-central nervous system tumor xenografts with an excellent safety profile.

Adenocarcinoma↗