SV40 Vectors Research Articles

Migration of bone marrow progenitor cells in the adult brain of rats and rabbits.

Neurogenesis takes place in the adult mammalian brain in three areas: Subgranular zone of the dentate gyrus (DG); subventricular zone of the lateral ventricle; olfactory bulb. Different molecular markers can be used to characterize the cells involved in adult neurogenesis. It has been recently suggested that a population of bone marrow (BM) progenitor cells may migrate to the brain and differentiate into neuronal lineage. To explore this hypothesis, we injected recombinant SV40-derived vectors into the BM and followed the potential migration of the transduced cells. Long-term BM-directed gene transfer using recombinant SV40-derived vectors leads to expression of the genes delivered to the BM firstly in circulating cells, then after several months in mature neurons and microglial cells, and thus without central nervous system (CNS) lesion. Most of transgene-expressing cells expressed NeuN, a marker of mature neurons. Thus, BM-derived cells may function as progenitors of CNS cells in adult animals. The mechanism by which the cells from the BM come to be neurons remains to be determined. Although the observed gradual increase in transgene-expressing neurons over 16 mo suggests that the pathway involved differentiation of BM-resident cells into neurons, cell fusion as the principal route cannot be totally ruled out. Additional studies using similar viral vectors showed that BM-derived progenitor cells migrating in the CNS express markers of neuronal precursors or immature neurons. Transgene-positive cells were found in the subgranular zone of the DG of the hippocampus 16 mo after intramarrow injection of the vector. In addition to cells expressing markers of mature neurons, transgene-positive cells were also positive for nestin and doublecortin, molecules expressed by developing neuronal cells. These cells were actively proliferating, as shown by short term BrdU incorporation studies. Inducing seizures by using kainic acid increased the number of BM progenitor cells transduced by SV40 vectors migrating to the hippocampus, and these cells were seen at earlier time points in the DG. We show that the cell membrane chemokine receptor, CCR5, and its ligands, enhance CNS inflammation and seizure activity in a model of neuronal excitotoxicity. SV40-based gene delivery of RNAi targeting CCR5 to the BM results in downregulating CCR5 in circulating cells, suggesting that CCR5 plays an important role in regulating traffic of BM-derived cells into the CNS, both in the basal state and in response to injury. Furthermore, reduction in CCR5 expression in circulating cells provides profound neuroprotection from excitotoxic neuronal injury, reduces neuroinflammation, and increases neuronal regeneration following this type of insult. These results suggest that BM-derived, transgene-expressing, cells can migrate to the brain and that they become neurons, at least in part, by differentiating into neuron precursors and subsequently developing into mature neurons.

Role of Oxidative Stress in HIV-1-Associated Neurocognitive Disorder and Protection by Gene Delivery of Antioxidant Enzymes.

HIV encephalopathy covers a range of HIV-1-related brain dysfunction. In the Central Nervous System (CNS), it is largely impervious to Highly Active AntiRetroviral Therapy (HAART). As survival with chronic HIV-1 infection improves, the number of people harboring the virus in their CNS increases. Neurodegenerative and neuroinflammatory changes may continue despite the use of HAART. Neurons themselves are rarely infected by HIV-1, but HIV-1 infects resident microglia, periventricular macrophages, leading to increased production of cytokines and to release of HIV-1 proteins, the most likely neurotoxins, among which are the envelope glycoprotein gp120 and HIV-1 trans-acting protein Tat. Gp120 and Tat induce oxidative stress in the brain, leading to neuronal apoptosis/death. We review here the role of oxidative stress in animal models of HIV-1 Associated Neurocognitive Disorder (HAND) and in patients with HAND. Different therapeutic approaches, including clinical trials, have been used to mitigate oxidative stress in HAND. We used SV40 vectors for gene delivery of antioxidant enzymes, Cu/Zn superoxide dismutase (SOD1), or glutathione peroxidase (GPx1) into the rat caudate putamen (CP). Intracerebral injection of SV (SOD1) or SV (GPx1) protects neurons from apoptosis caused by subsequent inoculation of gp120 and Tat at the same location. Vector administration into the lateral ventricle or cisterna magna protects from intra-CP gp120-induced neurotoxicity comparably to intra-CP vector administration. These models should provide a better understanding of the pathogenesis of HIV-1 in the brain as well as offer new therapeutic avenues.

Traitement laser Nd:YAG Long Pulse des veines superficielles de la main. Recherche d’un aspect esthétique irréprochable

We have analyzed the pronase-derived glycopeptides of the hemagglutinin glycoproteins expressed from SV40 vectors carrying cloned cDNA copies of the HA gene and of HA isolated from influenza virions (A/Jap/305/57). The glycopeptides derived from the HA glycoprotein obtained from cloned genes were heterogeneous, ranging in size from 3800 to 2800 dallons. Upon treatment with neuraminidase, sialic acid was released from the glycopeptides and their size was reduced to 2900–2400 daltons. However, under the same conditions, no sialic acid was detected in the virion HA. The presence of sialic acid was confirmed by monosaccharide analysis of the HA glycoprotein derived from products of cloned genes. These results support the idea that during replication of influenza virus, the viral neuraminidase cleaves sialic acid from the HA glycoprotein in infected cells.

HIV-1 Tat neurotoxicity: A model of acute and chronic exposure, and neuroprotection by gene delivery of antioxidant enzymes

HIV-associated neurocognitive disorder (HAND) is an increasingly common, progressive disease characterized by neuronal loss and progressively deteriorating CNS function. HIV-1 gene products, particularly gp120 and Tat elicit reactive oxygen species (ROS) that lead to oxidant injury and cause neuron apoptosis. Understanding of, and developing therapies for, HAND requires accessible models of the disease. We have devised experimental approaches to studying the acute and chronic effects of Tat on the CNS. We studied acute exposure by injecting recombinant Tat protein into the caudate-putamen (CP). Ongoing Tat expression, which more closely mimics HIV-1 infection of the brain, was studied by delivering Tat-expression over time using an SV40-derived gene delivery vector, SV(Tat). Both acute and chronic Tat exposure induced lipid peroxidation and neuronal apoptosis. Finally, prior administration of recombinant SV40 vectors carrying antioxidant enzymes, copper/zinc superoxide dismutase (SOD1) or glutathione peroxidase (GPx1), protected from Tat-induced apoptosis and oxidative injury. Thus, injection of recombinant HIV-1 Tat and the expression vector, SV(Tat), into the rat CP cause respectively acute or ongoing apoptosis and oxidative stress in neurons and may represent useful animal models for studying the pathogenesis and, potentially, treatment of HIV-1 Tat-related damage.

Intracisternal rSV40 administration provides effective pan-CNS transgene expression

Potential genetic treatments for many generalized central nervous system (CNS) diseases require transgene expression throughout the CNS. Using oxidant stress and apoptosis caused by HIV-1 envelope gp120 as a model, we studied pan-CNS neuroprotective gene delivery into the cisterna magna (CM). Recombinant SV40 vectors carrying Cu/Zn superoxide dismutase or glutathione peroxidase were injected into rat CMs following intraperitoneal administration of mannitol. Sustained transgene expression was seen in neurons throughout the CNS. On challenge, 8 weeks later with gp120 injected into the caudate putamen, significant neuroprotection was documented. Thus, intracisternal administration of antioxidant-carrying rSV40 vectors may be useful in treating widespread CNS diseases such as HIV-1-associated neurocognitive disorders characterized by oxidative stress.

A Rat Model of Human Immunodeficiency Virus 1 Encephalopathy Using Envelope Glycoprotein gp120 Expression Delivered by SV40 Vectors

Human immunodeficiency virus 1 (HIV-1) encephalopathy is thought to result in part from the toxicity of HIV-1 envelope glycoprotein gp120 for neurons. Experimental systems for studying the effects of gp120 and other HIV proteins on the brain have been limited to the acute effects of recombinant proteins in vitro or in vivo in simian immunodeficiency virus-infected monkeys. We describe an experimental rodent model of ongoing gp120-induced neurotoxicity in which HIV-1 envelope is expressed in the brain using an SV40-derived gene delivery vector, SV(gp120). When it is inoculated stereotaxically into the rat caudate putamen, SV(gp120) caused a partly hemorrhagic lesion in which neuron and other cell apoptosis continues for at least 12 weeks. Human immunodeficiency virus gp120 is expressed throughout this time, and some apoptotic cells are gp120 positive. Malondialdehyde and 4-hydroxynonenal assays indicated that there was lipid peroxidation in these lesions. Prior administration of recombinant SV40 vectors carrying antioxidant enzymes, copper/ zinc superoxide dismutase or glutathione peroxidase, was protective against SV(gp120)-induced oxidative injury and apoptosis. Thus, in vivo inoculation of SV(gp120) into the rat caudate putamen causes ongoing oxidative stress and apoptosis in neurons and may therefore represent a useful animal model for studying the pathogenesis and treatment of HIV-1 envelope-related brain damage.

219. Gene Delivery of Anti-CCR3 and Anti-CCR5 Trangenes Using SV40 Vectors Protect Infection of Monocyte-Derived Macrophages and Brain Microglia: Complexity in Co-Receptors Usage

219. Gene Delivery of Anti-CCR3 and Anti-CCR5 Trangenes Using SV40 Vectors Protect Infection of Monocyte-Derived Macrophages and Brain Microglia: Complexity in Co-Receptors Usage

Protecting neurons from HIV-1 gp120-induced oxidant stress using both localized intracerebral and generalized intraventricular administration of antioxidant enzymes delivered by SV40-derived vectors

Human immunodeficiency virus-1 (HIV-1) is the most frequent cause of dementia in adults under 40. We sought to use gene delivery to protect from HIV-1-related neuron loss. Because HIV-1 envelope (Env) gp120 elicits oxidant stress and apoptosis in cultured neurons, we established reproducible parameters of Env-mediated neurotoxicity in vivo, then tested neuroprotection using gene delivery of antioxidant enzymes. We injected 100-500 ng mul(-1)gp120 stereotaxically into rat caudate-putamens (CP) and assayed brains for apoptosis by terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling (TUNEL) 6-h to 14-day post-injection. Peak apoptosis occurred 1 day after injection of 250 and 500 ng microl(-1)gp120. TUNEL-positive cells mostly expressed neuronal markers (NeuroTrace), although some expressed CD68 and so were most likely microglial cells. Finally, we compared neuroprotection from gp120-induced apoptosis provided by localized and generalized intra-central nervous system (CNS) gene delivery. Recombinant SV40 vectors carrying Cu/Zn superoxide dismutase (SOD1) or glutathione peroxidase (GPx1) were injected into the CP, where gp120 was administered 4-24 weeks later. Alternatively, we inoculated the vector into the lateral ventricle (LV), with or without prior intraperitoneal (i.p.) administration of mannitol. Intracerebral injection of SV(SOD1) or SV(GPx1) significantly protected neurons from gp120-induced apoptosis throughout the 24-week study. Intraventricular vector administration protected from gp120 neurotoxicity comparably, particularly if preceded by mannitol i.p. Thus, HIV-1 gp120 is neurotoxic in vivo, and intracerebral or intra-ventricular administration of rSV40 vectors carrying antioxidant enzymes is neuroprotective. These findings suggest the potential utility of both localized and widespread gene delivery in treating neuroAIDS and other CNS diseases characterized by excessive oxidative stress.

988. Antioxidant Enzymes Delivered Using SV40 Vectors Protect Primary Neurons In Vitro from Apoptosis Mediated by Transduced HIV-1 gp120

988. Antioxidant Enzymes Delivered Using SV40 Vectors Protect Primary Neurons In Vitro from Apoptosis Mediated by Transduced HIV-1 gp120

Replication-deficient rSV40 mediate pancreatic gene transfer and long-term inhibition of tumor growth

Pancreatic cancer is one of the most aggressive and devastating human malignancies. There is an urgent need for more effective therapy for patients with advanced disease. In this context, genetic therapy potentially represents a rational new approach to treating pancreatic cancer, which could provide an adjunct to conventional options. Because of the promise of recombinant SV40 vectors, we tested their ability to deliver a transgene, and to target a transcript, so as to inhibit pancreatic tumors growth in vivo. BxPC3 and Capan-1 cells were efficiently transduced using SV40 vectors without selection, as compared to synthetic vectors PEI. SV40 vectors were as efficient as adenoviral vectors, and provided long-term transgene expression. Next, we devised a SV40-derived, targeted gene therapy approach of pancreatic cancer, by combining hTR tumor-specific promoter with sst2 somatostatin receptor tumor-suppressor gene. In vitro cell proliferation was strongly impaired following administration of SV(hTR-sst2). SV40-derived sst2-mediated antiproliferative effect was dependent on the local production of somatostatin. In vivo, intratumoral gene transfer of sst2 using rSV40 vectors resulted in a marked inhibition of Capan-1 tumor progression, and proliferation. These results represent the initial steps toward a novel approach to the gene therapy of pancreatic cancer using SV40 as a vector.

Antioxidant enzyme gene delivery to protect from HIV-1 gp120-induced neuronal apoptosis

Human immunodeficiency virus-1 (HIV-1) infection in the central nervous system (CNS) may lead to neuronal loss and progressively deteriorating CNS function: HIV-1 gene products, especially gp120, induce free radical-mediated apoptosis. Reactive oxygen species (ROS), are among the potential mediators of these effects. Neurons readily form ROS after gp120 exposure, and so might be protected from ROS-mediated injury by antioxidant enzymes such as Cu/Zn-superoxide dismutase (SOD1) and/or glutathione peroxidase (GPx1). Both enzymes detoxify oxygen free radicals. As they are highly efficient gene delivery vehicles for neurons, recombinant SV40-derived vectors were used for these studies. Cultured mature neurons derived from NT2 cells and primary fetal neurons were transduced with rSV40 vectors carrying human SOD1 and/or GPx1 cDNAs, then exposed to gp120. Apoptosis was measured by terminal deoxynucleotidyl transferase-mediated nick end labeling (TUNEL) assay. Transduction efficiency of both neuron populations was >95%, as assayed by immunostaining. Transgene expression was also ascertained by Western blotting and direct assays of enzyme activity. Gp120 induced apoptosis in a high percentage of unprotected NT2-N. Transduction with SV(SOD1) and SV(GPx1) before gp120 challenge reduced neuronal apoptosis by >90%. Even greater protection was seen in cells treated with both vectors in sequence. Given singly or in combination, they protect neuronal cells from HIV-1-gp120 induced apoptosis. We tested whether rSV40 s can deliver antioxidant enzymes to the CNS in vivo: intracerebral injection of SV(SOD1) or SV(GPx1) into the caudate putamen of rat brain yielded excellent transgene expression in neurons. In vivo transduction using SV(SOD1) also protected neurons from subsequent gp120-induced apoptosis after injection of both into the caudate putamen of rat brain. Thus, SOD1 and GPx1 can be delivered by SV40 vectors in vitro or in vivo. This approach may merit consideration for therapies in HIV-1-induced encephalopathy.

SV40 Pseudovirion gene delivery of a toxin to treat human adenocarcinomas in mice

SV40 vectors packaged in vitro (pseudovirions) are an efficient delivery system for plasmids up to 17.7 kb, with or without SV40 sequences. A truncated Pseudomonas exotoxin gene (PE38) was delivered into various human cells (HeLa, KB-3-1, human lymphoblastoids, and erythroleukemia cells), in vitro using pseudovirions. The number of viable cells was reduced significantly in the PE38-transduced cells. Human KB adenocarcinomas growing in mice were treated with intratumoral injection of PE38 packaged in vitro, and tumor size decreased significantly. Intraperitoneal treatments were as effective in reducing tumor size as intratumoral treatments. To check the viability of mock- or PE38-treated mice, every 4 days they were weighed, their blood was tested, and various tissues were screened for pathology. All parameters showed that the in vitro-packaged vectors, injected into tumors or intraperitoneally, caused no abnormalities in mice. The combined treatment of doxorubicin with in vitro-packaged PE38 reduced tumor size slightly more than each of the treatments separately. However, the combined treatment did not cause the weight loss seen with doxorubicin alone. These results indicate that SV40 in vitro packaging is an effective system for cancer gene delivery using two different routes of injection and in combination with chemotherapy.

Using gene delivery to protect HIV-susceptible CNS cells: Inhibiting HIV replication in microglia

Antiretroviral chemotherapy penetrates the CNS poorly. CNS HIV, thus sheltered, may injure the brain and complicate control of systemic HIV infection. Microglial cells play a major role in HIV persistence in the CNS but are rarely targeted for gene delivery. Because recombinant SV40 vectors (rSV40s) transduce other phagocytic cells efficiently, we tested rSV40 delivery of anti-HIV genetic therapy to microglial cells. Microglia prepared as enriched cultures from human fetal brain, were transduced with marker vectors, SV(RFP) and SV(Nef/FLAG), respectively, carrying DsRed and HIV-1 Nef bearing a FLAG epitope. By immunostaining and FACS, 95% of unselected cells expressed the transgenes, without detectable toxicity. Microglia were transduced with SV(AT), carrying human α1-antitrypsin (α1AT), which blocks Env and Gag processing. SV(AT)-treated microglia strongly resisted challenge with HIV-1BaL, even when microglia were transduced with SV(AT) following HIV challenge. Thus, rSV40s effectively transduce microglia and protect them from HIV.

584. Enhanced Inhibition of HIV Using Combinatorial Genetic Therapy Against HIV-1 Integrase Delivered by SV40 Vectors

Highly active anti-retroviral therapy, HAART, is a well-established combination pharmacotherapy. Its fundamental principle is to combat HIV-1 replication simultaneously at multiple points. However, the toxicity of HAART, its highly cost and rapid development of multiply resistant strains of HIV-1 all suggest that other therapeutic approaches are needed.

Long-term gene expression in dividing and nondividing cells using SV40-derived vectors.

Among the goals of gene therapy is long-term expression of delivered transgenes. Recombinant Tagdeleted SV40 vectors (rSV40s) are especially well suited for this purpose. rSV40s deliver transgene expression that endures for extended periods of time in tissue culture and in vivo, in both dividing and nondividing cells. These vectors are particularly effective in transducing some cell types that have been almost unapproachable using other gene delivery systems, such as quiescent hematopoietic progenitor cells and their differentiated derivatives. Other cellular targets include neurons, brain microglia, hepatocytes, dendritic cells, vascular endothelium, and others. Because rSV40s do not elicit neutralizing antibodies they are useful for in vivo gene delivery in settings where more than one administration may be desirable. The key characteristics of these vectors include their high production titers and therefore suitability for large cell pools, effectiveness in delivering intracellular proteins, and untranslated RNAs, maintenance of transgene expression at constant levels for extended times, suitability for constitutive or conditional promoters and for combinatorial gene delivery and ability to integrate into genomes of both dividing and nondividing cells.

896. Long Term Transgene Expression in the Brain Delivered by SV40-Derived Vectors

Top of pageAbstract Gene transfer to the central nervous system (CNS) has been studied using various vectors. Recombinant, Tag-deleted SV40 vectors provide long-term transgene expression in vitro and in vivo in multiple non-CNS cell types, and can be used in vitro to transduce both NT2-derived and primary human neurons, and primary human brain microglia. We previously reported administering a rSV40 vector into the caudate-putamen (CP) of the rat brain using stereotaxic conditions. This vector carries HIV-1 Nef protein with a carboxyl-terminal FLAG epitope tag driven with a CMV-IEP promoter, SV(Nef-FLAG). Transgene expression was assessed 4, 7 or 14 days later by immunostaining of serial coronal cryostat sections. Numerous transgene-positive cells were seen at those times, at least several mm longitudinally away from the injection site. The present study aimed to determine whether these vectors could also deliver long term expression of a transgene, up to 3 months after unilateral injection into the CP, hippocampus, and cerebellum (lobe 6). The uninjected side, or saline injection, were used as controls. No cells on the uninjected side were immunopositive for FLAG.Sections incubated with non-immune primary antibody (immunostain negative controls) were also negative. The number of transgene-positive cells counted 1, 2 and 3 months after injection of the vector in the CP was higher than the one at d14, and showed a wider anatomic distribution. Transgene-positive cells were seen not only in the CP, but also in different brain structures along the needle track. The longitudinal extent of the transduced area was similar along the whole CP for time points greater than 4 days. In the CP, many transgene-expressing cells stained positively for NeuN, or MAP2, indicating a substantial neuronal transduction, with more than 95% of the transduced cells co-immunostaining with these neuronal markers. Astrocytes generally did not express the transgene. Long term transgene expression was also seen in the hippocampus (dentate gyrus) and in the cerebellum. More transgene-positive cells were observed when 10 microl of vector were given than with an injection of 1 microl. In the cerebellum, transgene-positive cells colocalized with calbindin, a marker for Purkinje cells. Previous studies of rSV40 gene delivery did not identify inflammatory or immunologic responses to the vector. In these studies, we also looked for evidence of inflammatory or immune reaction to the transduction process, using sections stained with neutral red (NR): no inflammation was seen at any time point in brains injected in any of the different structures. Likewise, no CD8-positive T cells were identified in sections of brains injected with SV(Nef-FLAG). These results show that SV40-derived vectors can be used to achieve long term and efficient CNS gene transfer without evidence of inflammatory or immune reaction, preferentially to neurons and to a lesser extent, microglial cells, when injected in different parenchymal structures of the rat brain.

661. Efficient SV40-Mediated Gene Transfer to the Brain

Top of pageAbstract Gene transfer to the central nervous system has been accomplished with various vectors. SV40 vectors are able to transduce NT2-derived neurons and microglia in vitro. The goal of the present study was to determine the most effective routes of administration of SV40 vectors to the brain and the parameters of the transduction (diffusion, and distribution of the transgene in the brain as well as number and type of the transduced cells). As a marker, we used a rSV40 viral vector carrying HIV Nef protein with a carboxyl-terminal FLAG epitope tag-SV40(Nef-FLAG)- driven with a CMV-IEP promoter. The vector was injected using stereotaxic conditions into the lateral ventricle (LV) or, as an example of an intra-parenchymal inoculation site, the caudate-putamen (CP) of the rat brain. 4, 7, or 14 days later, transgene expression was assessed by immunocytochemistry on serial coronal cryostat sections. After injection of SV40 (Nef-FLAG) into the LV, FLAG expression was mainly observed in cells in the ependyma and choroid plexus. Numerous transgene-positive cells were observed 4, 7, and 14 days after the injection of SV40 (Nef-FLAG) into the CP. Seven days after the injection, transgene-positive cells were observed at least 4 mm longitudinally away from the injection site, and the average number of transgene-positive cells per hemisphere was 1202. In the CP, many transgene-expressing cells stained positively for NeuN, or MAP2, indicating a substantial neuronal transduction, with more than 95% of the transduced cells co-immunostaining with these neuronal markers. Numerous neurons were transduced in the vicinity of the needle track, but also far from the site of injection. There was no detectable expression of FLAG in the substantia nigra (SN), suggesting that retrograde transport of SV40 (Nef-FLAG) or the Nef FLAG protein from the CP to the SN does not play an important role in rSV40 gene delivery to CNS cells. These results show that SV40 vectors can be used to achieve an efficient gene transfer in neuronal cells when injected in the Rat CP, and to a lesser extent in the ependyma when injected into the LV.

965. Antioxidant Enzyme Protection from HIV-1 gp120-Induced Neuronal Apoptosis by Gene Delivery Using rSV40 Vectors: Implication for Gene Therapy of AIDS

Human immunodeficiency virus-1 (HIV-1) infection causes neuronal toxicity and oxidative injury: HIV-1 gene products, especially gp120, induce free radical-mediated apoptosis. Nitric oxide (NO), when present in high concentrations, is a potential mediator of neuronal injury via upregulation of inducible nitric oxide synthase (iNOS) and formation of reactive oxygen species (ROS).We thus tested whether rSV40 gene delivery to central nervous system (CNS) can be exploited to protect the brain and mitigate HIV encephalopathy. We hypothesized that neurons, which readily form ROS as a result of HIV-1/gp120 exposure, could be protected using rSV40 viruses encoding Cu/Zn-superoxide dismutase (SOD1) and/or glutathione peroxidase (GPX1). Both of these enzymes detoxify oxygen free radical species. Our test system was cultured mature neurons derived from NT-2 cells. These were transduced with rSV40 vectors carrying SOD1 or GPX1, or control rSV40 vectors, and challenged by adding HIV-1 gp120. Apoptosis was measured by TUNEL assay. Transduction with SV(SOD1) and SV(GPX1) provided excellent transgene expression when tested in terminally differentiated NT-2 neurons, as assessed by western blotting, kinetic assays of enzyme activity and immunostaining. Transduction efficiency by the recombinant SV40 delivered vectors exceeded 95% as assayed by immunostaining. When used on NT2 derived neurons, gp120, induced apoptosis with a linear dose response relationship with an optimal concentration of 100 ng/ml (0.9 nm) for induction of apoptosis. SV(SOD1) and SV(GPX1) transduced cells, when challenged with this dose of gp120, were protected up to >90% from apoptosis, as tested using TUNEL assay. Protection was dose dependent: suboptimal transducing doses of these vectors protected less well. Combination transduction with both viruses (SOD1 and GPX1), showed >95% protection against gp120 apoptosis. We therefore demonstrate the effectiveness of SOD1 and GPX1 delivered by SV40 vectors, either singly or in combination, to protect neuronal cells susceptible to HIV-1. These findings may help to address issues related to gene delivery to the central nervous system and gene therapy to protect from the encephalopathy that attends HIV-1 infection in brain.

659. The Plasticity of Hematopoietic Stem Cells (HSC): Rat HSC Transduced In Situ by rSV40 Vectors Differentiate into Multiple Lineages of CNS Cells

An increasing number of reports suggest that hematopoietic stem cells can migrate into the brain and differentiate into cell types only present in the CNS. In situ gene transfer to hematopoietic stem/progenitor cells by intramarrow injection of viral vectors has rarely been rarely reported and the consequent distribution of transgene-positive derivative cells in different organs has never been assessed. We injected recombinant Tag-deleted SV40 vectors (rSV40s) into the femoral bone marrow of rats. Test rats were given a rSV40 bearing a marker transgene, the FLAG epitope appended to the carboxyl terminus of HIV-1 Nef, which was used as a carrier protein. Control animals received an unrelated rSV40. FLAG expression was detected by FACS or fluorescence microscopy following intracellular immunostaining. Up to 12% (average, 5%) of circulating peripheral blood cells expressed the transgene through the end of the experiment at 18 months post injection. The distribution of FLAG-positive cells in the brain was extensively studied using serial cryostat sections of the brain taken at different times (< 1 month, 4 months, and 18 months) post-intramarrow injection. FLAG-positive cells were mainly found in the dentate gyrus (DG) of the hippocampus, either 4 or 18 months after the injection of SV40 (Nef-FLAG). Transgene-positive cells were found in the hilus, as well as in the inner/outer blades of the DG, with similar percentages for the 2 areas (5.2, and 5.5% of the total number of cells respectively). No expression of FLAG was seen in the brains of the control animals. Analysis of DG sections taken within 1 month of intra-marrow injection showed no evidence of such positive cells, indicating that direct transduction of the brain did not occur and suggesting that the FLAG-positive cells seen were the result of migration into the DG of transduced cells or their derivatives. The nature of the transgene-positive cells was assessed by double immunofluorescence using markers for neurons (neuN), microglia (ED1), and astrocytes (GFAP). The percentages of FLAG-positive cells expressing neuN, ED1, and GFAP, were respectively 48.6, 49.7, and 1.6%. FLAG expression was rarely seen in the few proliferating cells stained for PCNA. These data suggest that hematopoietic stem cells, or their derivatives, can migrate into the brain and differentiate into cells specific for the CNS.

Liver-Targeted Gene Therapy by SV40-Based Vectors Using the Hydrodynamic Injection Method

Efficient reconstitution of defective genes in hepatocytes could be used to treat various liver and systemic diseases through gene therapy. To explore the potential of SV40-based vectors in liver gene therapy, we constructed SV/luc, an SV40 T-antigen replacement transduction vector, that was propagated on COS and COT cells, which supply the SV40 T-antigen in trans. For liver targeting, BALB/C mice were injected via the tail vein with SV/luc stocks containing 3 x 10(6) to 10(8) transducing units in a volume of 1-2 ml. Luciferase activity was monitored with a light-detection cooled charged-coupled device (CCCD) camera, which enables continuous in vivo measurement of luc expression. The SV40 vector proved to be efficient in gene delivery to the liver, leading to long-term (> or =107 days) transgene expression in hepatocytes. Optimal results were obtained with 3 x 10(6) to 3 x 10(7) transducing units. The hydrodynamic vector delivery method caused transient liver inflammatory changes, with full recovery within days. Low levels of SV40-neutralizing antibodies were detected in the sera of treated mice; however, there was no indication of vector or transgene-specific cellular immune responses. Vectors packaged in vitro, using recombinant capsid proteins and plasmid DNA, were also effective in liver transduction. These results suggest that SV40 vectors may be useful for liver gene therapy.