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Eftekharpour E, Shcholok T. Cre-recombinase systems for induction of neuron-specific knockout models: a guide for biomedical researchers. Neural Regen Res 2023; 18:273-279. [PMID: 35900402 PMCID: PMC9396489 DOI: 10.4103/1673-5374.346541] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/04/2022] Open
Abstract
Gene deletion has been a valuable tool for unraveling the mysteries of molecular biology. Early approaches included gene trapping and gene targetting to disrupt or delete a gene randomly or at a specific location, respectively. Using these technologies in mouse embryos led to the generation of mouse knockout models and many scientific discoveries. The efficacy and specificity of these approaches have significantly increased with the advent of new technology such as clustered regularly interspaced short palindromic repeats for targetted gene deletion. However, several limitations including unwanted off-target gene deletion have hindered their widespread use in the field. Cre-recombinase technology has provided additional capacity for cell-specific gene deletion. In this review, we provide a summary of currently available literature on the application of this system for targetted deletion of neuronal genes. This article has been constructed to provide some background information for the new trainees on the mechanism and to provide necessary information for the design, and application of the Cre-recombinase system through reviewing the most frequent promoters that are currently available for genetic manipulation of neurons. We additionally will provide a summary of the latest technological developments that can be used for targeting neurons. This may also serve as a general guide for the selection of appropriate models for biomedical research.
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Choudhury SR, Hudry E, Maguire CA, Sena-Esteves M, Breakefield XO, Grandi P. Viral vectors for therapy of neurologic diseases. Neuropharmacology 2017; 120:63-80. [PMID: 26905292 PMCID: PMC5929167 DOI: 10.1016/j.neuropharm.2016.02.013] [Citation(s) in RCA: 123] [Impact Index Per Article: 15.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/19/2015] [Revised: 02/07/2016] [Accepted: 02/15/2016] [Indexed: 12/21/2022]
Abstract
Neurological disorders - disorders of the brain, spine and associated nerves - are a leading contributor to global disease burden with a shockingly large associated economic cost. Various treatment approaches - pharmaceutical medication, device-based therapy, physiotherapy, surgical intervention, among others - have been explored to alleviate the resulting extent of human suffering. In recent years, gene therapy using viral vectors - encoding a therapeutic gene or inhibitory RNA into a "gutted" viral capsid and supplying it to the nervous system - has emerged as a clinically viable option for therapy of brain disorders. In this Review, we provide an overview of the current state and advances in the field of viral vector-mediated gene therapy for neurological disorders. Vector tools and delivery methods have evolved considerably over recent years, with the goal of providing greater and safer genetic access to the central nervous system. Better etiological understanding of brain disorders has concurrently led to identification of improved therapeutic targets. We focus on the vector technology, as well as preclinical and clinical progress made thus far for brain cancer and various neurodegenerative and neurometabolic disorders, and point out the challenges and limitations that accompany this new medical modality. Finally, we explore the directions that neurological gene therapy is likely to evolve towards in the future. This article is part of the Special Issue entitled "Beyond small molecules for neurological disorders".
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Affiliation(s)
- Sourav R Choudhury
- Department of Neurology, University of Massachusetts Medical School, Worcester, MA 01605, USA; Gene Therapy Center, University of Massachusetts Medical School, Worcester, MA 01605, USA.
| | - Eloise Hudry
- Alzheimer's Disease Research Unit, Harvard Medical School & Massachusetts General Hospital, Charlestown, MA 02129, USA.
| | - Casey A Maguire
- Department of Neurology and Center for Molecular Imaging Research, Department of Radiology, Massachusetts General Hospital and NeuroDiscovery Center, Harvard Medical School, Boston, MA 02114, USA.
| | - Miguel Sena-Esteves
- Department of Neurology, University of Massachusetts Medical School, Worcester, MA 01605, USA; Gene Therapy Center, University of Massachusetts Medical School, Worcester, MA 01605, USA.
| | - Xandra O Breakefield
- Department of Neurology and Center for Molecular Imaging Research, Department of Radiology, Massachusetts General Hospital and NeuroDiscovery Center, Harvard Medical School, Boston, MA 02114, USA.
| | - Paola Grandi
- Department of Neurological Surgery, University of Pittsburgh Cancer Institute, Pittsburgh, PA 15219, USA.
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Dennie D, Louboutin JP, Strayer DS. Migration of bone marrow progenitor cells in the adult brain of rats and rabbits. World J Stem Cells 2016; 8:136-157. [PMID: 27114746 PMCID: PMC4835673 DOI: 10.4252/wjsc.v8.i4.136] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 06/02/2015] [Revised: 09/11/2015] [Accepted: 02/16/2016] [Indexed: 02/06/2023] Open
Abstract
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.
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Role of Oxidative Stress in HIV-1-Associated Neurocognitive Disorder and Protection by Gene Delivery of Antioxidant Enzymes. Antioxidants (Basel) 2014; 3:770-97. [PMID: 26785240 PMCID: PMC4665507 DOI: 10.3390/antiox3040770] [Citation(s) in RCA: 23] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/13/2014] [Revised: 10/26/2014] [Accepted: 10/28/2014] [Indexed: 12/26/2022] Open
Abstract
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.
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Louboutin JP, Agrawal L, Reyes BAS, van Bockstaele EJ, Strayer DS. Gene delivery of antioxidant enzymes inhibits human immunodeficiency virus type 1 gp120-induced expression of caspases. Neuroscience 2012; 214:68-77. [PMID: 22531373 DOI: 10.1016/j.neuroscience.2012.03.061] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/21/2011] [Revised: 03/14/2012] [Accepted: 03/16/2012] [Indexed: 01/03/2023]
Abstract
Caspases are implicated in neuronal death in neurodegenerative and other central nervous system (CNS) diseases. In a rat model of human immunodeficiency virus type 1 (HIV-1) associated neurocognitive disorders (HAND), we previously characterized HIV-1 envelope gp120-induced neuronal apoptosis by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. In this model, neuronal apoptosis occurred probably via gp120-induced reactive oxygen species (ROS). Antioxidant gene delivery blunted gp120-related apoptosis. Here, we studied the effect of gp120 on different caspases (3, 6, 8, 9) expression. Caspases production increased in the rat caudate-putamen (CP) 6h after gp120 injection into the same structure. The expression of caspases peaked by 24h. Caspases colocalized mainly with neurons. Prior gene delivery of the antioxidant enzymes Cu/Zn superoxide dismutase (SOD1) or glutathione peroxidase (GPx1) into the CP before injecting gp120 there reduced levels of gp120-induced caspases, recapitulating the effect of antioxidant enzymes on gp120-induced apoptosis observed by TUNEL. Thus, HIV-1 gp120 increased caspases expression in the CP. Prior antioxidant enzyme treatment mitigated production of these caspases, probably by reducing ROS levels.
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Affiliation(s)
- J-P Louboutin
- Department of Pathology, Anatomy and Cell Biology, Thomas Jefferson University, Philadelphia, PA 19107, United States.
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HIV-1 Tat neurotoxicity: a model of acute and chronic exposure, and neuroprotection by gene delivery of antioxidant enzymes. Neurobiol Dis 2011; 45:657-70. [PMID: 22036626 DOI: 10.1016/j.nbd.2011.10.005] [Citation(s) in RCA: 55] [Impact Index Per Article: 3.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/29/2011] [Revised: 09/14/2011] [Accepted: 10/08/2011] [Indexed: 11/21/2022] Open
Abstract
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.
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Louboutin JP, Reyes BAS, Agrawal L, Van Bockstaele EJ, Strayer DS. Intracisternal rSV40 administration provides effective pan-CNS transgene expression. Gene Ther 2011; 19:114-8. [PMID: 21614027 DOI: 10.1038/gt.2011.75] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/29/2023]
Abstract
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.
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Affiliation(s)
- J-P Louboutin
- Department of Pathology, Anatomy and Cell Biology, Thomas Jefferson University, Philadelphia, PA, USA.
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Louboutin JP, Reyes BAS, Van Bockstaele EJ, Strayer DS. Gene transfer to the cerebellum. THE CEREBELLUM 2011; 9:587-97. [PMID: 20700772 DOI: 10.1007/s12311-010-0202-9] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/22/2022]
Abstract
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.
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Affiliation(s)
- Jean-Pierre Louboutin
- Department of Pathology, Jefferson Medical College, Thomas Jefferson University, 1020 Locust Street, Room 255, Philadelphia, PA 19107, USA.
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Louboutin JP, Marusich E, Fisher-Perkins J, Dufour JP, Bunnell BA, Strayer DS. Gene transfer to the rhesus monkey brain using SV40-derived vectors is durable and safe. Gene Ther 2011; 18:682-91. [DOI: 10.1038/gt.2011.13] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/09/2022]
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Louboutin JP, Chekmasova AA, Marusich E, Chowdhury JR, Strayer DS. Efficient CNS gene delivery by intravenous injection. Nat Methods 2010; 7:905-7. [PMID: 20953176 DOI: 10.1038/nmeth.1518] [Citation(s) in RCA: 42] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/19/2010] [Accepted: 09/13/2010] [Indexed: 12/24/2022]
Abstract
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.
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Affiliation(s)
- Jean-Pierre Louboutin
- Department of Pathology, Thomas Jefferson University, Philadelphia, Pennsylvania, USA.
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Meland MN, Herndon ME, Stipp CS. Expression of alpha5 integrin rescues fibronectin responsiveness in NT2N CNS neuronal cells. J Neurosci Res 2010; 88:222-32. [PMID: 19598247 PMCID: PMC2863016 DOI: 10.1002/jnr.22171] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/10/2022]
Abstract
The extracellular matrix protein fibronectin is implicated in neuronal regeneration in the peripheral nervous system. In the central nervous system (CNS), fibronectin is up-regulated at sites of penetrating injuries and stroke; however, CNS neurons down-regulate the fibronectin receptor alpha5beta1 integrin during differentiation and generally respond poorly to fibronectin. NT2N CNS neuron-like cells (derived from NT2 precursor cells) have been used in preclinical and clinical studies for treatment of stroke and a variety of CNS injury and disease models. Here we show that, like primary CNS neurons, NT2N cells down-regulate alpha5beta1 integrin during differentiation and respond poorly to fibronectin. The poor neurite outgrowth by NT2N cells on fibronectin can be rescued by transducing NT2 precursors with a retroviral vector expressing alpha5 integrin under the control of the murine stem cell virus 5' long terminal repeat. Sustained alpha5 integrin expression is compatible with the CNS-like neuronal differentiation of NT2N cells and does not prevent robust neurite outgrowth on other integrin ligands. Thus, alpha5 integrin expression in CNS neuronal precursor cells may provide a strategy for enhancing the outgrowth and survival of implanted cells in cell-replacement therapies for CNS injury and disease.
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Affiliation(s)
- Marit N Meland
- Department of Biology, University of Iowa, Iowa City, Iowa, USA
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A strongly transdominant mutation in the human immunodeficiency virus type 1 gag gene defines an Achilles heel in the virus life cycle. J Virol 2009; 83:8536-43. [PMID: 19515760 DOI: 10.1128/jvi.00317-09] [Citation(s) in RCA: 69] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/25/2022] Open
Abstract
The human immunodeficiency virus type 1 (HIV-1) protease (PR) makes five obligatory cleavages in the viral Gag polyprotein precursor. The cleavage events release the virion structural proteins from the precursor and allow the virion to undergo maturation to become infectious. The protease cleavage between the matrix protein (MA) domain and the adjacent capsid protein (CA) domain releases CA from the membrane-anchored MA and allows the N terminus of CA to refold into a structure that facilitates the formation of hexamer arrays that represent the structural unit of the capsid shell. In this study, we analyzed the extent to which each of the HIV-1 Gag processing sites must be cleaved by substituting the P1-position amino acid at each processing site with Ile. A mutation that blocks cleavage at the MA/CA processing site (Y132I) displayed a strong transdominant effect when tested in a phenotypic mixing strategy, inhibiting virion infectivity with a 50% inhibitory concentration of only 4% of the mutant relative to the wild type. This mutation is 10- to 20-fold more potent in phenotypic mixing than an inactivating mutation in the viral protease, the target of many successful inhibitors, and more potent than an inactivating mutation at any of the other Gag cleavage sites. The transdominant effect is manifested as the assembly of an aberrant virion core. Virus containing 20% of the Y132I mutant and 80% of the wild type (to assess the transdominant effect on infectivity) was blocked either before reverse transcription (RT) or at an early RT step. The ability of a small amount of the MA/CA fusion protein to poison the oligomeric assembly of infectious virus identifies an essential step in the complex process of virion formation and maturation. The effect of a small-molecule inhibitor that is able to block MA/CA cleavage even partially would be amplified by this transdominant negative effect on the highly orchestrated process of virion assembly.
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Louboutin JP, Agrawal L, Reyes BAS, Van Bockstaele EJ, Strayer DS. HIV-1 gp120 neurotoxicity proximally and at a distance from the point of exposure: protection by rSV40 delivery of antioxidant enzymes. Neurobiol Dis 2009; 34:462-76. [PMID: 19327399 DOI: 10.1016/j.nbd.2009.03.003] [Citation(s) in RCA: 20] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/26/2008] [Revised: 02/26/2009] [Accepted: 03/04/2009] [Indexed: 01/21/2023] Open
Abstract
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.
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Affiliation(s)
- Jean-Pierre Louboutin
- Department of Pathology, Anatomy, and Cell Biology, Thomas Jefferson University, Philadelphia, PA 19107, USA.
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Legiewicz M, Badorrek CS, Turner KB, Fabris D, Hamm TE, Rekosh D, Hammarskjöld ML, Le Grice SFJ. Resistance to RevM10 inhibition reflects a conformational switch in the HIV-1 Rev response element. Proc Natl Acad Sci U S A 2008; 105:14365-70. [PMID: 18776047 PMCID: PMC2567145 DOI: 10.1073/pnas.0804461105] [Citation(s) in RCA: 54] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/08/2008] [Indexed: 11/18/2022] Open
Abstract
Nuclear export of certain HIV-1 mRNAs requires an interaction between the viral Rev protein and the Rev response element (RRE), a structured element located in the Env region of its RNA genome. This interaction is an attractive target for both drug design and gene therapy, exemplified by RevM10, a transdominant negative protein that, when introduced into host cells, disrupts viral mRNA export. However, two silent G->A mutations in the RRE (RRE61) confer RevM10 resistance, which prompted us to examine RRE structure using a novel chemical probing strategy. Variations in region III/IV/V of mutant RNAs suggest a stepwise rearrangement to RevM10 resistance. Mass spectrometry was used to directly assess Rev "loading" onto RRE and its variants, indicating that this is unaffected by RNA structural changes. Similarity in chemical footprints with mutant protein implicates additional host factors in RevM10 resistance.
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Affiliation(s)
- Michal Legiewicz
- *HIV Drug Resistance Program, National Cancer Institute, Frederick, MD 21702
| | | | - Kevin B. Turner
- Department of Chemistry and Biochemistry, University of Maryland Baltimore County, Baltimore, MD 21250; and
| | - Daniele Fabris
- Department of Chemistry and Biochemistry, University of Maryland Baltimore County, Baltimore, MD 21250; and
| | - Tiffany E. Hamm
- Myles Thayler Center for AIDS and Human Retrovirus Research and Department of Microbiology, University of Virginia, Charlottesville, VA 22908
| | - David Rekosh
- Myles Thayler Center for AIDS and Human Retrovirus Research and Department of Microbiology, University of Virginia, Charlottesville, VA 22908
| | - Marie-Louise Hammarskjöld
- Myles Thayler Center for AIDS and Human Retrovirus Research and Department of Microbiology, University of Virginia, Charlottesville, VA 22908
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Louboutin JP, Agrawal L, Liu B, Strayer DS. In vivogene transfer to the CNS using recombinant SV40-derived vectors. Expert Opin Biol Ther 2008; 8:1319-35. [DOI: 10.1517/14712598.8.9.1319] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/31/2022]
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Louboutin JP, Agrawal L, Reyes BAS, Van Bockstaele EJ, Strayer DS. 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. Gene Ther 2007; 14:1650-61. [PMID: 17914406 DOI: 10.1038/sj.gt.3303030] [Citation(s) in RCA: 34] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/17/2023]
Abstract
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.
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Affiliation(s)
- J-P Louboutin
- Department of Pathology, Anatomy and Cell Biology, Thomas Jefferson University, Philadelphia, PA 19107, USA.
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Agrawal L, Louboutin JP, Strayer DS. Preventing HIV-1 tat-induced neuronal apoptosis using antioxidant enzymes: Mechanistic and therapeutic implications. Virology 2007; 363:462-72. [PMID: 17336361 DOI: 10.1016/j.virol.2007.02.004] [Citation(s) in RCA: 43] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/09/2007] [Accepted: 02/01/2007] [Indexed: 12/20/2022]
Abstract
HIV-1 proteins, especially gp120 and Tat, elicit reactive oxygen species (ROS) and cause neuron apoptosis. We used antioxidant enzymes, Cu/Zn superoxide dismutase (SOD1) and glutathione peroxidase (GPx1) to study signaling and neuroprotection from Tat-induced apoptosis. SOD1 converts superoxide to peroxide; GPx1 converts peroxide to water. Primary human neurons were transduced with SV40-derived vectors carrying SOD1 and GPx1, then HIV-1 Tat protein was added. Both SV(SOD1) and SV(GPx1) delivered substantial transgene expression. Tat decreased endogenous cellular, but not transduced, SOD1 and GPx1. Tat rapidly increased neuron [Ca(2+)](i), which effect was not altered by SV(SOD1) or SV(GPx1). However, both vectors together blocked Tat-induced [Ca(2+)](i) fluxes. Similarly, neither SV(SOD1) nor SV(GPx1) protected neurons from Tat-induced apoptosis, but both vectors together did. Tat therefore activates multiple signaling pathways, in one of which superoxide acts as an intermediate while the other utilizes peroxide. Gene delivery to protect neurons from Tat must therefore target both.
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Affiliation(s)
- Lokesh Agrawal
- Department of Pathology, Anatomy, and Cell Biology, Thomas Jefferson University, Room 251, 1020 Locust Street, Philadelphia, PA 19107, USA
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Louboutin JP, Reyes BAS, Agrawal L, Van Bockstaele E, Strayer DS. Strategies for CNS-directed gene delivery: in vivo gene transfer to the brain using SV40-derived vectors. Gene Ther 2007; 14:939-49. [PMID: 17443215 DOI: 10.1038/sj.gt.3302939] [Citation(s) in RCA: 32] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/21/2022]
Abstract
Gene transfer to the central nervous system (CNS) has been approached using various vectors. Recombinant SV40-derived vectors (rSV40s) transduce neurons and microglia effectively in vitro, so we tested rSV40s gene transfer to the CNS in vivo, and characterized the distribution, duration and cell types transduced. We used rSV40s carrying Human Immunodeficiency Virus Type 1 Net protein (HIV-1 Nef) with a C-terminal FLAG epitope tag as a marker, and another with Cu/Zn superoxide dismutase (SOD1). Rats were given vectors stereotaxically, either intraparenchymally into the caudate-putamen (CP) or into the lateral ventricle (LV). FLAG expression was studied for 3 months by immunostaining serial brain sections. After intraparenchymal administration, numerous transgene-expressing cells were seen, many as far as 4 mm from the injection site. Transgene expression remained strong throughout the 3-month study period. Coimmunostaining for lineage markers showed that neurons and, more rarely, microglial cells were tranduced, except astrocytes and oligodendroglia. After injection into the LV, high levels of transgene expression were detected throughout the frontal cortex by Western analysis. Systemic mannitol-induced hyperosmolarity further augmented LV transgene delivery. SV40-derived vectors may, thus, be useful for long-term gene expression in the brain, whether locally by intraparenchymal administration or diffusely by intraventricular injection, with or without mannitol.
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Affiliation(s)
- J-P Louboutin
- Department of Pathology, Anatomy and Cell Biology, Thomas Jefferson University, Philadelphia, PA 19107, USA.
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19
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Congote LF. Serpin A1 and CD91 as host instruments against HIV-1 infection: are extracellular antiviral peptides acting as intracellular messengers? Virus Res 2007; 125:119-34. [PMID: 17258834 DOI: 10.1016/j.virusres.2006.12.018] [Citation(s) in RCA: 24] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/03/2006] [Revised: 12/15/2006] [Accepted: 12/22/2006] [Indexed: 01/11/2023]
Abstract
Serpin A1 (alpha1-antitrypsin, alpha1-proteinase inhibitor) has been shown to be a non-cytolytic antiviral factor present in blood and effective against HIV infection. The best known physiological role of serpin A1 is to inhibit neutrophil elastase, a proteinase which is secreted by neutrophils at sites of infection and inflammation. Decreased HIV-infectivity is associated with decreased density of membrane-associated elastase. The enzyme may facilitate binding of the HIV membrane protein gp120 to host cells, and it specifically cleaves SDF-1, the physiological ligand of the HIV-1 co-receptor CXCR4. It has been suggested that one of the actions of serpin A1 as antiviral agent is to reduce HIV infectivity, and this property could be due to elastase inhibition. However, the most dramatic effect of serpin A1 is inhibition of HIV production. In vitro experiments indicate that the C-terminal peptide of serpin A1, produced during the formation of the complex of serpin with serine proteinases, may be responsible for the inhibition of HIV-1 expression in infected cells. This peptide, an integral part of the serpin-enzyme complex, is internalized by several scavenger receptors. Peptides corresponding to the C-terminal section of serpin A1 inhibit HIV-1 long-terminal-repeat-driven transcription and interact with nuclear proteins, such as alpha1-fetoprotein transcription factor. LDL-receptor-related protein 1 (LRP1/CD91), the best known receptor for serpin-enzyme complexes, is up-regulated in monocytes of HIV-1-infected true non-progressors. CD91 could be one of the major players in host resistance against HIV-1. It has the capacity of internalizing antiviral peptides such as serpin C-terminal fragments and alpha-defensins, and is at the same time the receptor for heat-shock proteins in antigen-presenting cells, in which chaperoned viral peptides could lead to the induction of cytotoxic T-cell responses.
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Affiliation(s)
- Luis Fernando Congote
- Endocrine Laboratory, McGill University Health Centre, 687 Avenue des pins, Ouest, Montreal, H3A 1A1, Canada.
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Cordelier P, Bienvenu C, Lulka H, Marrache F, Bouisson M, Openheim A, Strayer DS, Vaysse N, Pradayrol L, Buscail L. Replication-deficient rSV40 mediate pancreatic gene transfer and long-term inhibition of tumor growth. Cancer Gene Ther 2006; 14:19-29. [PMID: 16990845 DOI: 10.1038/sj.cgt.7700987] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/03/2023]
Abstract
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.
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Affiliation(s)
- P Cordelier
- INSERM U531, Institut Louis Bugnard, IFR31, Toulouse, France.
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Louboutin JP, Liu B, Reyes BAS, Van Bockstaele EJ, Strayer DS. Rat bone marrow progenitor cells transduced in situ by rSV40 vectors differentiate into multiple central nervous system cell lineages. Stem Cells 2006; 24:2801-9. [PMID: 16960137 DOI: 10.1634/stemcells.2006-0124] [Citation(s) in RCA: 21] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/24/2022]
Abstract
Using bone marrow-directed gene transfer, we tested whether bone marrow-derived cells may function as progenitors of central nervous system (CNS) cells in adult animals. SV40-derived gene delivery vectors were injected directly into femoral bone marrow, and we examined transgene expression in blood and brain for 0-16 months thereafter by immunostaining for FLAG epitope marker. An average of 5% of peripheral blood cells and 25% of femoral marrow cells were FLAG(+) throughout the study. CNS FLAG-expressing cells were mainly detected in the dentate gyrus (DG) and periventricular subependymal zone (PSZ). Although absent before 1 month and rare at 4 months, DG and PSZ FLAG(+) cells were abundant 16 months after bone marrow injection. Approximately 5% of DG cells expressed FLAG, including neurons (48.6%) and microglia (49.7%), and occasional astrocytes (1.6%), as determined by double immunostaining for FLAG and lineage markers. These data suggest that one or more populations of cells resident within adult bone marrow can migrate to the brain and differentiate into CNS-specific cells.
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Affiliation(s)
- Jean-Pierre Louboutin
- Department of Pathology, Anatomy, and Cell Biology, Jefferson Medical College, 1020 Locust Street, Philadelphia, Pennsylvania 19107, USA.
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22
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Agrawal L, Louboutin JP, Reyes BAS, Van Bockstaele EJ, Strayer DS. Antioxidant enzyme gene delivery to protect from HIV-1 gp120-induced neuronal apoptosis. Gene Ther 2006; 13:1645-56. [PMID: 16871233 DOI: 10.1038/sj.gt.3302821] [Citation(s) in RCA: 41] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/20/2022]
Abstract
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.
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Affiliation(s)
- L Agrawal
- Department of Pathology, Anatomy, and Cell Biology, Thomas Jefferson University, 1020 Locust Street, Philadelphia, PA 19107, USA.
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23
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Cordelier P, Strayer DS. Using gene delivery to protect HIV-susceptible CNS cells: inhibiting HIV replication in microglia. Virus Res 2006; 118:87-97. [PMID: 16414141 DOI: 10.1016/j.virusres.2005.11.016] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/09/2005] [Revised: 11/04/2005] [Accepted: 11/18/2005] [Indexed: 01/13/2023]
Abstract
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.
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Affiliation(s)
- Pierre Cordelier
- Department of Pathology, Jefferson Medical College, 1020 Locust Street, Room 251, Philadelphia, PA 19107, USA
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24
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Arad U, Zeira E, El-Latif MA, Mukherjee S, Mitchell L, Pappo O, Galun E, Oppenheim A. Liver-targeted gene therapy by SV40-based vectors using the hydrodynamic injection method. Hum Gene Ther 2005; 16:361-71. [PMID: 15812231 DOI: 10.1089/hum.2005.16.361] [Citation(s) in RCA: 41] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/12/2022] Open
Abstract
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.
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Affiliation(s)
- Uri Arad
- Department of Hematology, Hebrew University-Hadassah Medical School and Hadassah Hospital, Jerusalem 91120, Israel
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Liu B, Daviau J, Nichols CN, Strayer DS. In vivo gene transfer into rat bone marrow progenitor cells using rSV40 viral vectors. Blood 2005; 106:2655-62. [PMID: 15994284 PMCID: PMC1895314 DOI: 10.1182/blood-2005-01-0028] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/24/2022] Open
Abstract
Hematopoietic stem cell (HSC) gene transfer has been attempted almost entirely ex vivo and has been limited by cytokine-induced loss of self-renewal capacity and transplantation-related defects in homing and engraftment. Here, we attempted to circumvent such limitations by injecting vectors directly into the bone marrow (BM) to transduce HSCs in their native environment. Simian virus 40 (SV40)-derived gene delivery vectors were used because they transduce resting CD34+ cells very efficiently. Rats received SV-(Nef-FLAG), carrying FLAG marker epitope--or a control recombinant SV40 (rSV40)--directly into both femoral marrow cavities. Intracellular transgene expression by peripheral blood (PB) or BM cells was detected by cytofluorimetry. An average of 5.3% PB leukocytes expressed FLAG for the entire study--56 weeks. Transgene expression was sustained in multiple cell lineages, including granulocytes (average, 3.3% of leukocytes, 20.4% of granulocytes), CD3+ T lymphocytes (average, 0.53% of leukocytes, 1% of total T cells), and CD45R+ B lymphocytes, indicating gene transfer to long-lived progenitor cells with multilineage capacity. An average of 15% of femoral marrow cells expressed FLAG up to 16.5 months after transduction. Thus, direct intramarrow administration of rSV40s yields efficient gene transfer to rat BM progenitor cells and may be worthy of further investigation.
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Affiliation(s)
- Bianling Liu
- Department of Pathology, Jefferson Medical College, 1020 Locust St, Rm 251, Philadelphia, PA 19107, USA
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26
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Wolkowicz R, Nolan GP. Gene therapy progress and prospects: novel gene therapy approaches for AIDS. Gene Ther 2005; 12:467-76. [PMID: 15703764 DOI: 10.1038/sj.gt.3302488] [Citation(s) in RCA: 36] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/20/2022]
Abstract
Acquired immunodeficiency syndrome (AIDS), caused by human immunodeficiency virus (HIV), kills millions worldwide every year. Vaccines against HIV still seem a distant promise. Pharmaceutical treatments exist, but these are not always effective, and there is increasing prevalence of viral strains with multidrug resistance. Highly active antiretroviral therapy (HAART) consists of inhibitors of viral enzymes (reverse transcriptase (RT) and protease). Gene therapy, first introduced as intracellular immunization, may offer hopes for new treatments to be used alone, or in conjunction with, conventional small molecule drugs. Gene therapy approaches against HIV-1, including suicide genes, RNA-based technology, dominant negative viral proteins, intracellular antibodies, intrakines, and peptides, are the subject of this review.
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Affiliation(s)
- R Wolkowicz
- Department of Microbiology and Immunology, School of Medicine, Stanford University, Stanford, CA, USA
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28
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Vera M, Prieto J, Strayer DS, Fortes P. Factors Influencing the Production of Recombinant SV40 Vectors. Mol Ther 2004; 10:780-91. [PMID: 15451462 DOI: 10.1016/j.ymthe.2004.06.1014] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/15/2004] [Revised: 05/27/2004] [Accepted: 06/07/2004] [Indexed: 10/26/2022] Open
Abstract
Most gene therapy approaches employ viral vectors for gene delivery. Ideally, these vectors should be produced at high titer and purity with well-established protocols. Standardized methods to measure the quality of the vectors produced are imperative, as are techniques that allow reproducible quantitation of viral titer. We devised a series of protocols that achieve high-titer production and reproducible purification and provide for quality control and titering of recombinant simian virus 40 vectors (rSV40s). rSV40s are good candidate vehicles for gene transfer: they are easily modified to be nonreplicative and they are nonimmunogenic. Further, they infect a wide variety of cells and allow long-term transgene expression. We report here these protocols to produce rSV40 vectors in high yields, describe their purification, and characterize viral stocks using quality control techniques that monitor the presence of wild-type SV40 revertants and defective interfering particles. Several methods for reproducible titration of rSV40 viruses have been compared. We believe that these techniques can be widely applied to obtain high concentrations of high-quality rSV40 viruses reproducibly.
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Affiliation(s)
- Maria Vera
- Laboratory of Vector Development, Division of Gene Therapy, Foundation for Applied Medical Research, School of Medicine, University of Navarra, Irunlarrea 1, 31008 Pamplona, Spain
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Abstract
Simian virus-40 (SV40), an icosahedral papovavirus, has recently been modified to serve as a gene delivery vector. Recombinant SV40 vectors (rSV40) are good candidates for gene transfer, as they display some unique features: SV40 is a well-known virus, nonreplicative vectors are easy-to-make, and can be produced in titers of 10(12) IU/ml. They also efficiently transduce both resting and dividing cells, deliver persistent transgene expression to a wide range of cell types, and are nonimmunogenic. Present disadvantages of rSV40 vectors for gene therapy are a small cloning capacity and the possible risks related to random integration of the viral genome into the host genome. Considerable efforts have been devoted to modifing this virus and setting up protocols for viral production. Preliminary therapeutic results obtained both in tissue culture cells and in animal models for heritable and acquired diseases indicate that rSV40 vectors are promising gene transfer vehicles. This article reviews the work performed with SV40 viruses as recombinant vectors for gene transfer. A summary of the structure, genomic organization, and life cycle of wild-type SV40 viruses is presented. Furthermore, the strategies utilized for the development, production, and titering of rSV40 vectors are discussed. Last, the therapeutic applications developed to date are highlighted.
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Affiliation(s)
- Maria Vera
- School of Medicine, Foundation for Applied Medical Research, Division of Gene Therapy, Laboratory of Vectors Development, University of Navarra, Pamplona, Spain
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Davidson BL, Breakefield XO. Viral vectors for gene delivery to the nervous system. Nat Rev Neurosci 2003; 4:353-64. [PMID: 12728263 DOI: 10.1038/nrn1104] [Citation(s) in RCA: 245] [Impact Index Per Article: 11.1] [Reference Citation Analysis] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/14/2023]
Affiliation(s)
- Beverly L Davidson
- Program in Gene Therapy, Department of Internal Medicine, Roy J. and Lucille A. Carver College of Medicine, University of Iowa, Iowa City, Iowa 52242, USA.
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