RESUMO
We administered recombinant SV40-derived viral vectors (rSV40s) intravenously to mice with or without prior intraperitoneal injection of mannitol to deliver transgenes to the central nervous system (CNS). We detected transgene-expressing cells (mainly neurons) most prominently in the cortex and spinal cord; prior intraperitoneal mannitol injection increased CNS gene delivery tenfold. Intravenous injection of rSV40s, particularly with mannitol pretreatment, resulted in extensive expression of multiple transgenes throughout the CNS.
Assuntos
Córtex Cerebral/metabolismo , Técnicas de Transferência de Genes , Vírus 40 dos Símios/genética , Medula Espinal/metabolismo , Transgenes , Animais , Células COS , Chlorocebus aethiops , Terapia Genética , Injeções Intravenosas , Masculino , Camundongos , Camundongos Endogâmicos BALB CRESUMO
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.
Assuntos
Complexo AIDS Demência/terapia , Antioxidantes/administração & dosagem , Modelos Animais de Doenças , Terapia Genética/métodos , Infecções por HIV/terapia , HIV-1 , Produtos do Gene tat do Vírus da Imunodeficiência Humana/toxicidade , Animais , Apoptose/fisiologia , Feminino , Vetores Genéticos , Infecções por HIV/complicações , Imuno-Histoquímica , Marcação In Situ das Extremidades Cortadas , Peroxidação de Lipídeos/fisiologia , Fármacos Neuroprotetores/administração & dosagem , Ratos , Ratos Sprague-Dawley , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/toxicidade , Vírus 40 dos Símios/genética , Produtos do Gene tat do Vírus da Imunodeficiência Humana/administração & dosagemRESUMO
Chemokines may play a role in leukocyte migration across the blood-brain barrier (BBB) during neuroinflammation and other neuropathological processes, such as epilepsy. We investigated the role of the chemokine receptor CCR5 in seizures. We used a rat model based on intraperitoneal kainic acid (KA) administration. Four months before KA injection, adult rats were given femoral intramarrow inoculations of SV (RNAiR5-RevM10.AU1), which carries an interfering RNA (RNAi) against CCR5, plus a marker epitope (AU1), or its monofunctional RNAi-carrying homologue, SV(RNAiR5). This treatment lowered expression of CCR5 in circulating cells. In control rats, seizures induced elevated expression of CCR5 ligands MIP-1α and RANTES in the microvasculature, increased BBB leakage and CCR5(+) cells, as well as neuronal loss, inflammation, and gliosis in the hippocampi. Animals given either the bifunctional or the monofunctional vector were largely protected from KA-induced seizures, neuroinflammation, BBB damage, and neuron loss. Brain CCR5 mRNA was reduced. Rats receiving RNAiR5-bearing vectors showed far greater repair responses: increased neuronal proliferation, and decreased production of MIP-1α and RANTES. Controls received unrelated SV(BUGT) vectors. Decrease in CCR5 in circulating cells strongly protected from excitotoxin-induced seizures, BBB leakage, CNS injury, and inflammation, and facilitated neurogenic repair.
Assuntos
Quimiocina CCL3/metabolismo , Quimiocina CCL5/metabolismo , Ácido Caínico/toxicidade , Receptores CCR5/metabolismo , Convulsões/induzido quimicamente , Animais , Barreira Hematoencefálica/efeitos dos fármacos , Encéfalo/citologia , Encéfalo/efeitos dos fármacos , Quimiocina CCL3/genética , Quimiocina CCL5/genética , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Gliose , Hipocampo/citologia , Hipocampo/efeitos dos fármacos , Ligantes , Neurônios/citologia , Neurônios/efeitos dos fármacos , Neurônios/patologia , Neurotoxinas/toxicidade , Interferência de RNA , Ratos , Ratos Sprague-Dawley , Receptores CCR5/genética , Regeneração , Convulsões/prevenção & controleRESUMO
The blood-brain barrier (BBB) is compromised in many systemic and CNS diseases, including HIV-1 infection of the brain. We studied BBB disruption caused by HIV-1 envelope glycoprotein 120 (gp120) as a model. Exposure to gp120, whether acute [by direct intra-caudate-putamen (CP) injection] or chronic [using SV(gp120), an experimental model of ongoing production of gp120] disrupted the BBB, and led to leakage of vascular contents. Gp120 was directly toxic to brain endothelial cells. Abnormalities of the BBB reflect the activity of matrix metalloproteinases (MMPs). These target laminin and attack the tight junctions between endothelial cells and BBB basal laminae. MMP-2 and MMP-9 were upregulated following gp120-injection. Gp120 reduced laminin and tight junction proteins. Reactive oxygen species (ROS) activate MMPs. Injecting gp120 induced lipid peroxidation. Gene transfer of antioxidant enzymes protected against gp120-induced BBB abnormalities. NMDA upregulates the proform of MMP-9. Using the NMDA receptor (NMDAR-1) inhibitor, memantine, we observed partial protection from gp120-induced BBB injury. Thus, (1) HIV-envelope gp120 disrupts the BBB; (2) this occurs via lesions in brain microvessels, MMP activation and degradation of vascular basement membrane and vascular tight junctions; (3) NMDAR-1 activation plays a role in this BBB injury; and (4) antioxidant gene delivery as well as NMDAR-1 antagonists may protect the BBB.
Assuntos
Barreira Hematoencefálica , Proteína gp120 do Envelope de HIV/fisiologia , Animais , Membrana Basal/patologia , Encéfalo/irrigação sanguínea , Células Cultivadas , Endotélio Vascular/patologia , Glutationa Peroxidase/genética , HIV-1 , Humanos , Metaloproteinases da Matriz/biossíntese , Microvasos/patologia , Estresse Oxidativo/fisiologia , Receptores de N-Metil-D-Aspartato/metabolismo , Superóxido Dismutase/genética , Superóxido Dismutase-1 , Junções Íntimas/patologia , Glutationa Peroxidase GPX1RESUMO
Matrix metalloproteinases (MMPs) are implicated in diverse processes, such as neuroinflammation, leakiness of the blood-brain barrier (BBB) and direct cellular damage in neurodegenerative and other CNS diseases. Tissue destruction by MMPs is regulated by their endogenous tissue inhibitors (TIMPs). TIMPs prevent excessive MMP-related degradation of extracellular matrix components. In a rat model of human immunodeficiency virus (HIV)-related encephalopathy, we described MMP-2 and MMP-9 upregulation by HIV-1 envelope gp120, probably via gp120-induced reactive oxygen species. Antioxidant gene delivery blunted gp120-induced MMP production. We also studied the effect of gp120 on TIMP-1 and TIMP-2 production. TIMP-1 and TIMP-2 levels increased 6 h after gp120 injection into rat caudate-putamen (CP). TIMP-1 and TIMP-2 colocalized mainly with neurons (92 and 95%, respectively). By 24 h, expression of these protease inhibitors diverged, as TIMP-1 levels remained high but TIMP-2 subsided. Gene delivery of the antioxidant enzymes Cu/Zn superoxide dismutase or glutathione peroxidase into the CP before injecting gp120 there reduced levels of gp120-induced TIMP-1 and TIMP-2, recapitulating the effect of antioxidant enzymes on gp120-induced MMP-2 and MMP-9. A significant correlation was observed between MMP/TIMP upregulation and BBB leakiness. Thus, HIV-1 gp120 upregulated TIMP-1 and TIMP-2 in the CP. Prior antioxidant enzyme treatment mitigated production of these TIMPs, probably by reducing MMP expression.
Assuntos
Complexo AIDS Demência/fisiopatologia , Núcleo Caudado/efeitos dos fármacos , Proteína gp120 do Envelope de HIV/farmacologia , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Putamen/efeitos dos fármacos , Inibidor Tecidual de Metaloproteinase-1/metabolismo , Inibidor Tecidual de Metaloproteinase-2/metabolismo , Complexo AIDS Demência/patologia , Animais , Barreira Hematoencefálica/efeitos dos fármacos , Barreira Hematoencefálica/patologia , Barreira Hematoencefálica/fisiologia , Núcleo Caudado/citologia , Núcleo Caudado/fisiologia , Modelos Animais de Doenças , Feminino , Glutationa Peroxidase/genética , Glutationa Peroxidase/metabolismo , Proteína gp120 do Envelope de HIV/genética , Humanos , Putamen/citologia , Putamen/fisiologia , Ratos , Ratos Sprague-Dawley , Espécies Reativas de Oxigênio/metabolismo , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismo , Regulação para CimaRESUMO
HIV-1 effects on the blood-brain barrier (BBB) structure and function are still poorly understood in animal models based on direct administration of recombinant HIV proteins. We therefore injected HIV-1 envelope glycoprotein, gp120, into rat caudate-putamens (CPs) and examined vascular integrity and function. Gp120 coimmunostained with endothelial cell marker, CD31. It induced apoptosis of endothelial cells in vitro and in vivo. BBB function was assessed by administering Evans Blue (EB) intravenously before injecting gp120. EB leaked near the site of gp120 administration. Within 1h after intra-CP gp120 injection, structures positive for endothelial markers ICAM-1 and RECA-1 were greatly decreased. Vascular density assessed by laminin immunostaining remained decreased 1 month after gp120 injection. RECA-1-positive cells expressed hydroxynonenal, a marker of lipid peroxidation and rSV40-mediated gene delivery of antioxidant enzymes protected the BBB from gp120-related injury. Extravasated IgG accumulated following intra-CP SV(gp120) injection, an experimental model of continuing gp120 exposure. Thus: acute and chronic exposure to gp120 disrupts the BBB; gp120-mediated BBB abnormalities are related to lesions of brain microvessels; and gp120 is directly toxic to brain endothelial cells.
Assuntos
Barreira Hematoencefálica/metabolismo , Núcleo Caudado/metabolismo , Glutationa Peroxidase/genética , Proteína gp120 do Envelope de HIV/administração & dosagem , Superóxido Dismutase/genética , Animais , Antioxidantes , Apoptose/fisiologia , Barreira Hematoencefálica/patologia , Núcleo Caudado/patologia , Células Cultivadas , Feminino , Imunofluorescência , Técnicas de Transferência de Genes , Terapia Genética , Vetores Genéticos , Glutationa Peroxidase/administração & dosagem , Humanos , Marcação In Situ das Extremidades Cortadas , Neurônios/metabolismo , Neurônios/patologia , Estresse Oxidativo/fisiologia , Ratos , Ratos Sprague-Dawley , Estatísticas não Paramétricas , Superóxido Dismutase/administração & dosagemRESUMO
There are several diseases for which gene transfer therapy to the cerebellum might be practicable. In these studies, we used recombinant Tag-deleted SV40-derived vectors (rSV40s) to study gene delivery targeting the cerebellum. These vectors transduce neurons and microglia very effectively in vitro and in vivo, and so we tested them to evaluate gene transfer to the cerebellum in vivo. Using a rSV40 vector carrying human immunodeficiency virus (HIV)-Nef with a C-terminal FLAG epitope, we characterized the distribution, duration, and cell types transduced. Rats received test and control vectors by stereotaxic injection into the cerebellum. Transgene expression was assessed 1, 2, and 4 weeks later by immunostaining of serial brain sections. FLAG epitope-expressing cells were seen, at all times after vector administration, principally detected in the Purkinje cells of the cerebellum, identified as immunopositive for calbindin. Occasional microglial cells were tranduced; transgene expression was not detected in astrocytes or oligodendrocytes. No inflammatory or other reaction was detected at any time. Thus, SV40-derived vectors can deliver effective, safe, and durable transgene expression to the cerebellum.
Assuntos
Cerebelo/metabolismo , Transdução Genética/métodos , Animais , Contagem de Células/métodos , Cerebelo/citologia , Feminino , Expressão Gênica/fisiologia , Produtos do Gene nef/genética , Produtos do Gene nef/metabolismo , Vetores Genéticos/fisiologia , Proteínas do Tecido Nervoso/metabolismo , Neurônios/metabolismo , Ratos , Ratos Sprague-Dawley , Vírus 40 dos Símios/genética , Estatísticas não ParamétricasRESUMO
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.
Assuntos
Complexo AIDS Demência/etiologia , Modelos Animais de Doenças , Proteína gp120 do Envelope de HIV/efeitos adversos , Proteína gp120 do Envelope de HIV/metabolismo , HIV-1/patogenicidade , Complexo AIDS Demência/genética , Adenovirus dos Símios , Aldeídos/metabolismo , Animais , Proteínas de Ligação ao Cálcio , Morte Celular , Proteínas de Ligação a DNA/metabolismo , Feminino , Regulação Viral da Expressão Gênica/fisiologia , Vetores Genéticos/administração & dosagem , Proteína Glial Fibrilar Ácida/metabolismo , Glutationa Peroxidase/metabolismo , Humanos , Marcação In Situ das Extremidades Cortadas , Indóis , Proteínas dos Microfilamentos , Neurônios/patologia , Neurônios/virologia , Fosfopiruvato Hidratase/metabolismo , Putamen/virologia , Ratos , Ratos Nus , Ratos Sprague-Dawley , Estatísticas não Paramétricas , Fatores de Tempo , Transdução Genética/métodosRESUMO
Toxicity of HIV-1 envelope glycoprotein (gp120) for substantia nigra (SN) neurons may contribute to the Parkinsonian manifestations often seen in HIV-1-associated dementia (HAD). We studied the neurotoxicity of gp120 for dopaminergic neurons and potential neuroprotection by antioxidant gene delivery. Rats were injected stereotaxically into their caudate-putamen (CP); CP and (substantia nigra) SN neuron loss was quantified. The area of neuron loss extended several millimeters from the injection site, approximately 35% of the CP area. SN neurons, outside of this area of direct neurotoxicity, were also severely affected. Dopaminergic SN neurons (expressing tyrosine hydroxylase, TH, in the SN and dopamine transporter, DAT, in the CP) were mostly affected: intra-CP gp120 caused approximately 50% DAT+ SN neuron loss. Prior intra-CP gene delivery of Cu/Zn superoxide dismutase (SOD1) or glutathione peroxidase (GPx1) protected SN neurons from intra-CP gp120. Thus, SN dopaminergic neurons are highly sensitive to HIV-1 gp120-induced neurotoxicity, and antioxidant gene delivery, even at a distance, is protective.
Assuntos
Dopamina/metabolismo , Glutationa Peroxidase/metabolismo , Proteína gp120 do Envelope de HIV/toxicidade , Neurônios/fisiologia , Superóxido Dismutase/metabolismo , Animais , Núcleo Caudado/patologia , Núcleo Caudado/fisiopatologia , Morte Celular , Proteínas da Membrana Plasmática de Transporte de Dopamina/metabolismo , Feminino , Vetores Genéticos , Glutationa Peroxidase/genética , Imuno-Histoquímica , Ratos , Ratos Sprague-Dawley , Vírus 40 dos Símios/genética , Substância Negra/patologia , Substância Negra/fisiopatologia , Superóxido Dismutase/genética , Superóxido Dismutase-1 , Tirosina 3-Mono-Oxigenase/metabolismoRESUMO
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 alpha1-antitrypsin (alpha1AT), 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.
Assuntos
Terapia Genética/métodos , HIV-1/fisiologia , Microglia/virologia , Replicação Viral , Células Cultivadas , Ensaio de Imunoadsorção Enzimática , Vetores Genéticos , Proteína do Núcleo p24 do HIV/análise , HIV-1/genética , Humanos , Vírus 40 dos Símios/genética , Transdução Genética , alfa 1-Antitripsina/genéticaRESUMO
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.
Assuntos
Expressão Gênica , Terapia Genética/métodos , Vetores Genéticos/genética , Vírus 40 dos Símios/genética , Vírus 40 dos Símios/imunologia , Animais , Divisão Celular , Técnicas de Transferência de Genes , Terapia Genética/efeitos adversos , Humanos , Mutagênese Insercional , Integração ViralRESUMO
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.
RESUMO
Proprotein processing is essential for HIV infectivity. Cellular trans-Golgi network (TGN) serine proteases (e.g., furin) are required to cleave HIV envelope gp160 to gp120. In addition, HIV protease (PR), an aspartyl protease, cleaves p55(Gag) to p24, etc., in budding virions. alpha1-Antitrypsin (alpha(1)AT) is cleaved by serine proteases, causing a conformational change in alpha(1)AT that sequesters and so inactivates the protease. alpha(1)AT blocks both gp160 and p55 processing, and so is a powerful inhibitor of HIV replication. We hypothesized that alpha(1)AT inhibited gp160 and p55 processing via different mechanisms, and that in both cases, alpha(1)AT bound and was itself cleaved by the proteases whose activities were blocked. alpha(1)AT delivered by SV(AT), a recombinant, Tag-deleted SV40-derived vector, localized to the TGN, co-precipitated with furin, and depleted furin from the TGN. After SV(AT) transduction and HIV challenge, alpha(1)AT was detected in resulting nascent immature HIV-1 virions. alpha(1)AT also blocked incorporation of the enzymatically active dimeric form of PR into HIV virions. Western analysis using recombinant proteins showed that alpha(1)AT directly bound HIV PR, and was cleaved by it. The simultaneous inhibition of two different steps in HIV morphogenesis both increases alpha(1)AT antilentiviral activity and decreases the possibility that HIV mutations will allow escape from inhibition.
Assuntos
Inibidores da Protease de HIV/metabolismo , Protease de HIV/metabolismo , HIV-1/fisiologia , Subtilisinas/antagonistas & inibidores , Linfócitos T/virologia , alfa 1-Antitripsina/metabolismo , Animais , Células COS , Furina , Complexo de Golgi/enzimologia , Proteína gp160 do Envelope de HIV/metabolismo , Protease de HIV/química , HIV-1/metabolismo , Células HeLa , Humanos , Conformação Proteica , Subtilisinas/análise , Linfócitos T/enzimologia , Linfócitos T/metabolismo , Transdução Genética , alfa 1-Antitripsina/química , alfa 1-Antitripsina/genéticaRESUMO
The natural function of viruses is to deliver their genetic material to cells. Among the most effective of viruses in doing that is Simian Virus-40 (SV40). The properties that make SV40 a successful virus make it an attractive candidate for use as a gene delivery vehicle: high titer replication, infectivity for almost all nucleated cell types whether the cells are dividing or resting, potential for integration into cellular DNA, a peculiar pathway for entering cells that bypasses the cells' antigen processing apparatus, very high stability, and the apparent ability to activate expression of its own capsid genes in trans. Exploiting these and other characteristics of wild type (wt) SV40, increasing numbers of laboratories are studying recombinant (r) SV40-derived vectors. Among the uses to which these vectors have been applied are: delivering therapy to inhibit HIV, hepatitis C virus (HCV) and other viruses; correction of inherited hepatic and other protein deficiencies; immunizing against lentiviral and other antigens; treatment of inherited and acquired diseases of the central nervous system; protecting the lung and other organs from free radical-induced injury; and many others. The effectiveness of these vectors is a reflection of the adaptive evolution that produced their parent virus, wt SV40. This article explores how and why these vectors work, their strengths and their limitations, and provides a functional model for their exploitation for experimental and clinical applications.
Assuntos
Terapia Genética , Vetores Genéticos , Vírus 40 dos Símios/genética , Animais , Humanos , Transdução Genética , Transgenes , Replicação ViralRESUMO
The rapid increase in the study of small interfering RNA (siRNA) as a means to decrease expression of targeted genes has led to concerns about possible unexpected consequences of constitutive siRNA expression. We therefore devised a conditional siRNA expression system in which siRNA targeting hepatitis C virus (HCV) would be produced in response to HCV. We found that HCV acts via NFkappaB to stimulate the HIV long terminal repeat (LTR) as a promoter. We exploited this observation by designing conditional siRNA transcription constructs to be triggered by HCV-induced activation of NFkappaB. These were delivered by using highly efficient recombinant Tag-deleted SV40-derived vectors. Conditional activation of HIV-LTR and consequent siRNA synthesis in cells expressing HCV were observed. HCV-specific RNAi decreased HCV RNA greatly within 4 days, using transient transfection of the whole HCV genome as a model of acute HCV entry into transduced cells. We then tested the effectiveness of rSV40-delivered anti-HCV siRNA in cells stably transfected with the whole HCV genome to simulate hepatocytes chronically infected with HCV. There is considerable need for regulated production of siRNAs activated by a particular set of conditions (HCV in this case) but quiescent otherwise. Approaches described here may serve as a paradigm for such conditional siRNA expression.
Assuntos
Interferência de RNA , Animais , Sequência de Bases , Northern Blotting , Linhagem Celular , Primers do DNA , DNA Complementar , Hepacivirus/genética , Plasmídeos , RNA Viral/análise , RNA Viral/genética , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Chronic infection with hepatitis B virus (HBV) has potentially devastating consequences and is very difficult to treat. Therapy with recombinant interferons (IFN), especially IFN-alpha, may be effective. The blood IFN-alpha levels that are needed to maintain therapeutic IFN-alpha levels in the liver, however, often cause severe side effects. Gene delivery to the liver may provide a solution. Using a long-term expression construct could provide the desired levels of IFN locally without the need to maintain potentially problematic blood levels. Recombinant, Tag-deleted SV40-derived vectors transduce hepatocytes efficiently and provide permanent transgene expression. We designed an expression construct that was effective against HBV and whose activity was limited to HBV-infected cells. To do this, we exploited the ability of HBV X protein to activate NF-kappaB and, via NF-kappaB, to activate promoter activity of HIV long terminal repeat (LTR) in hepatocytes. Using HIVLTR as a conditional promoter upstream of human and murine IFN-alpha and IFN-gamma cDNAs, rSV40 vectors were used to test the responsiveness of IFN to HBV and the ability of these IFNs to inhibit HBV transcripts and protein production and to activate IFN signaling in neighboring untransduced cells. We found that in hepatocyte cell lines and in primary hepatocytes, HBV activated the promoter activity of the HIVLTR via NF-kappaB. When whole HBV genome was delivered to cells by transfection to simulate HBV infection, IFN expression was activated, IFNs were produced and secreted, and they protected cells from HBV. Levels of IFN proteins that were secreted in this context were comparable to targeted blood levels needed to control chronic hepatitis viral infection. Further, IFNs that were elicited and secreted in this manner were able to activate IFN-induced signaling pathways in neighboring, untransduced cells and so were likely to provide protection even to cells that the rSV40 vector did not transduce. Gene delivery using such rSV40 vectors expressing IFNs conditionally in response to HBV may be an attractive therapeutic option for the treatment of chronic hepatitis B.
Assuntos
Terapia Genética/métodos , Vírus da Hepatite B/fisiologia , Hepatite B/genética , Hepatite B/terapia , Interferon-alfa/metabolismo , Interferon gama/metabolismo , Animais , Linhagem Celular Tumoral , Células Cultivadas , Regulação da Expressão Gênica , Repetição Terminal Longa de HIV/genética , Haplorrinos , Hepatite B/metabolismo , Hepatite B/patologia , Hepatócitos/metabolismo , Hepatócitos/virologia , Humanos , Interferon-alfa/genética , Interferon gama/genética , Fígado/metabolismo , Fígado/patologia , Camundongos , Regiões Promotoras Genéticas/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Elementos de Resposta/genética , Transdução de Sinais , Vírus 40 dos Símios/genéticaRESUMO
In human immunodeficiency virus type 1 (HIV-1)-infected individuals, virus-induced production of interferon alpha (IFN-alpha) is impaired. In order to obtain regulated expression of IFN-alpha that responds to HIV-1 infection, a recombinant SV40 vector was designed that carries the human IFN-alpha2 cDNA under the control of the HIV-1 long terminal repeat (LTR) (SV[HIVLTR]IFN). Thus, the IFN-alpha2 gene would be trans-activated on infection with HIV-1. This vector was tested to determine if central nervous system (CNS) cell types that may be potential HIV-1 targets could be transduced and protected from HIV. SV[HIVLTR]IFN transduced NT2 cells, a human neuronal precursor cell line, mature neurons derived from NT2 precursor cells, and human primary monocyte-derived macrophages. IFN-alpha2 expression was retained in mature neurons after SV[HIVLTR]IFN-transduced NT2 precursor cells were induced to differentiate using retinoic acid. IFN-alpha expression was detected only after exposing transduced cells to HIV. Furthermore, SV[HIVLTR]IFN-delivered IFN-alpha2 expression significantly inhibited replication of multiple strains of HIV in both NT2 and NT2-derived mature neurons. SV[HIVLTR]IFN transduction also inhibited HIV-1(BaL) replication in human primary monocyte-derived macrophages. Therefore, we have demonstrated the effectiveness of IFN-alpha2, delivered by an SV40 vector driven by HIV-1 LTR as a promoter, to protect several CNS-based, potentially HIV-susceptible cell types. These findings may have implications for therapy of HIV-1 infection in the CNS.
Assuntos
Terapia Genética , Vetores Genéticos , Infecções por HIV/terapia , HIV-1/efeitos dos fármacos , Interferon Tipo I/genética , Vírus 40 dos Símios , Doenças do Sistema Nervoso Central/terapia , Humanos , Interferon Tipo I/metabolismo , Proteínas RecombinantesRESUMO
Transducing macrophages and other phagocytic cells has been problematic because these cells are largely nondividing and can phagocytose and degrade viral gene delivery vectors. Because of their carriage of the CCR5 chemokine receptor that functions as a coreceptor for most clinical strains of HIV, these cells are also key targets in early HIV infection and dissemination. We describe here a strategy to transduce these phagocytes, reduce cell membrane CCR5, and protect from infection with R5-tropic HIV. Recombinant Tag-deleted SV40 vectors were used to transduce unselected CCR5-bearing cell lines and primary cells with >98% efficiency. rSV40s were designed to express two different anti-CCR5 small interfering RNAs (siRNAs), driven by the adenoviral VA1 polymerase III (pol III) promoter, which localizes the transcripts in the cytoplasm. Transduction with both siRNAs substantially reduced CCR5 mRNA, which in turn decreased detectable cell membrane CCR5. Both CCR5+ cell lines and primary cells were used: SupT1/CCR5 cells, monocyte-derived macrophages (MDM), and primary human brain microglia. In addition, one siRNA, siRNA R5 #5, was designed to recognize conserved sequences in both murine and human CCR5 mRNA and effectively reduced CCR5 transcript in cells of both species. These siRNAs largely protected CCR5+ cell lines and primary human macrophages and brain microglia from challenge with R5-tropic HIV. Therefore, strategies to target CCR5 using rSV40-delivered, VA promoter-driven siRNAs may be useful therapeutic options for treating HIV infection.
Assuntos
HIV-1/fisiologia , Macrófagos/imunologia , Macrófagos/virologia , Microglia/imunologia , RNA Interferente Pequeno/genética , Receptores CCR5/genética , Vírus 40 dos Símios/genética , Animais , Encéfalo/citologia , Células COS , Diferenciação Celular , Linhagem Celular , Chlorocebus aethiops , Clonagem Molecular , Feto , Regulação da Expressão Gênica/imunologia , Vetores Genéticos , Humanos , Macrófagos/citologia , Camundongos , Microglia/virologia , Monócitos/citologia , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Deleção de Sequência , TransfecçãoRESUMO
The limited success of gene therapy as an approach to treating human disease largely reflects the limitations of the gene delivery vectors that have been used. Poor titers, low transduction efficiency, waning transgene expression and immunogenicity have remained obstacles in the field. As a consequence, much research in normal, immunocompetent animals has not demonstrated therapeutic levels of gene delivery, and results from most human clinical trials have been predictably discouraging. Recombinant gene transfer vectors derived from SV40 virus (rSV40) are potentially of great interest for those working in gene therapy, since these vectors are not subject to many of the problems that have limited gene delivery using other vector systems. rSV40 is made at a very high titer and infects - and so transduces - almost all nucleated cell types very efficiently, regardless of lineage or whether they are resting or dividing; they integrate and are not susceptible to transgene silencing; and they elicit no detectable immune response on the part of normal animals and so can be used to deliver multiple transgenes over time and in sequence. The recent development of 'gutless' rSV40 vectors has expanded the range of potential therapeutic transgenes that can be delivered with this system and added flexibility to the expression configurations that can be accommodated. All of these functional characteristics of SV40-derived vectors have their bases in the biology of SV40 and similar viruses, and have important implications for the potential utility of rSV40 vectors in gene therapeutics. Like all viral gene delivery systems, these vectors have their idiosyncrasies and limitations. They also allow gene delivery that bypasses many of the difficulties that have plagued the field from its inception.