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532. Trans-Synaptic Neurotoxicity of HIV-1 gp120 and Trans-Synaptic Protection Using rSV40 Vectors Delivery Antioxidant Enzymes
531. Cell Targeting in the CNS Using HIV-1Based Lentiviral Vectors Bearing Alternative Glycoproteins and Promoters
Thais Federici,' Robert H. Kutner.? Noel Tordo.' Jun Yang,' Erin Carlton,' Nicholas M. Boulis,' Jakob Reiser,' I Neurosciences, Cleve/and Clinic, Cleveland, OH; 2 Departm ent ofGene Therapy. Louisiana State University, Nell' Orleans, LA; "Lyssavirus Laboratory, Institut Pasteur; Paris, France .
Background: AAV and Rabies G-pseudotyped EIAV-based lentiviral vectors are the most promising vectors in the field of gene transfer and potential candidates for clinical applications for ncurodegenerative diseases. Lentiviral vectors derived from EIAV and HIV·I have twice the genomic capacity of AAV vectors, which allows insertion of additional transgenesequencesthat may improve their therapeutic outcome. The delivery of therapeutic proteins to the CNS representsan appealingstrategy for the treatmentof nerve injuryand disordersofthe CNS.lmportant factorsdeterminingCNS targeting includetropismand retrogradetransportof the vector particles. Herewe comparednovelpseudotyped HIV-I-basedlentiviral vectors, evaluating their ability to transduce specific cell populations and promote retrograde axonal transport in vitro. Methods: HIV-I-based vectors, pseudotyped with the G glycoproteins from dilTerent enveloped viruses belongingto the Rhabdoviridae family, genus Lyssavirus (RabiesG, European Bat, Lagos Bat, Duvenhage, VSV-G), were used. The vectors expressed the Enhanced Green Fluorescent Protein (EGFP) or the E. coli J3-galactosidase (LacZ) transgcncs, controlled by the constitutiveCytomegalovirus(CMVIE) promoter. Wetested their performancefor efficientdeliveryand expression of transgenes in NSC-34 cells and rat primary mixed spinal cord cultures. We then analyzed their uptake and retrograde transport in vitro, using Campenot chambers. We also constructed and tested VSV-G pseudotypedvectors,expressingthe EGFPtransgene and bearingdifferentpromotersincludingthe humanubiquitin C (hUbC) promoterand the human synapsin-I (hSYNI) promoter. Flowcytometry(FACS), X-galstaining,and immunocytochemistry were performed. Results: We had previously performed a in vitro study with SH-SY5Y cells and demonstrated that our Rabies Gpseudotyped HIV-I-based vector possessed the highest neuronal tropismamongthe pseudotypestested. In thisseries of experiments, the Rabies G-pseudotyped vector demonstrated again the highest levelsofJ3-Gal-positive cells in mixed spinal cord cultures,as well as the best uptake and retrogradetransport in Campenotchambers. Concerningthe promoters,all the vectors showed robust transgene expression in NSC-34 cells as judged by flow cytometry, The transgene expression with the hUbc and hSYNI promoters was strong and exclusivelyneuronal, whereas the transgeneexpression from the hCMV-IE promoter was robust, but also present in glial cells, as shown by immunocytochemistry. Conclusion: Our in vitro studies demonstrated that the Rabies G-pseudotyped vector possessed the best performance and neuronal tropism among the pseudotypes tested in vitro. Because the pattern of transduction in vitro docs not necessarily reflect the uptake in vivo, additional studiesarc underwayto confirmthe transductionpropertiesofthese vectors using an animal model. Acknowledgement : This research was supported by grants from NINDS K08 NS43305, ALSA and NINDS ROI NS44832.
532. Trans-Synaptic Neurotoxicity of HIV-1 gp120 and Trans-Synaptic Protection Using rSV40 Vectors Delivery Antioxidant Enzymes
Jean-Pierre Louboutin,' LokeshAgrawal; BeverlyA. S. Reyes,' ElisabethJ. van Bockstaele,' David S. Strayer,' I Pathology; Thomas Jefferson University, Philadelphia, PA; 'Neurosurgery and Farber Institute for Neurosciences, Thomas Jefferson University; Philadelphia, PA.
The basalgangliaare oftenaffectedin patientswith HIV-I-associated dementia (I·IAD). In addition to signs of neuron loss in many areas, Parkinson-like symptomatology is common in people with HAD.The neurotoxicityof HIV-I envelopeglycoprotein, gp120,is thoughtto be in partresponsible for the neuronlossin HADpatients. We developed an animal model to study gpl20-induced lesions in the rat brain by injectingvaryingdoses of gp120stereotaxically into the caudate-putamen (CP), then determining the extent of neuron loss. We then tested the ability of antioxidant gene transfer using recombinant SV40-derived vectors delivering CulZn superoxide dismutase (SV(SOD1» or glutathione peroxidase (SV(GPxl». Neuron loss was measured by TUNEL assay for apoptosis and by enumerating neurotrace-positive cells.Whenwe injected gp120(100 ng, 250 ng or 500 ng) into the CP, tissue damage extended several microns rostral and caudal to the injectionsite, as observed on neutral red (NR)-stainedcryostat sections 7 and 14days after injection. The area of the damage quantified by morphometric analysis on Nk-stained sections was about 25% of the total area of the Cp' 7 and 14days after injectionof500 nggpl20. (100 ng gpl20 induced minimal lesions). Staining of neurons by Neurotraee (NT) showed a loss of 30% of Nl-positivc cells during the same time period. Similar results were found on sections immunostained for neuN, a marker of neurons. However. neuronal loss was also observed in dilTerent cortical areas at 7 and 14 days after intra-CP injection of gp120. Specifically, in the striatum many neurons are involved in dopaminergic systemsand expressthe dopaminetransporter(OAT). Numbers of OAT-positive cells were decreased by 50% at 7 and 14 days after injectionof gp120 in the CPo A decrease in the number of tyrosine-hydroxylase (n-I)-positive neurons was also observed in the SN, the principal efTeetor of the dopaminergic system, from 4 to 14 days after injectionof gp120 in the CPo This finding suggests that gp120-induced neurotoxicity could extend beyond one and two synapses from the site of exposure. Although the mechanism by which this occurs is not clear, in another abstract, we document axonal transport of gp120. Furthermore, injection of SV(SOOI) or SV(GPxl) into the CP, from 4 to 24 weeks before injection of 500 ng gp120 in the Cp' significantlyand substantiallyreduced the extent of the injury, the loss of NT- and OAT-positive cells in the cr, as well as the diminution of the Tll-positive cells in the SN. Therefore, we demonstratethat the neurotoxicity ofl-IIV-I envelope gp120 can traverse synapses and that the protection afforded by prior gene transferof SODI or GPxI using rSV40 vectors may also traverse synapses.These findingsbear both on the pathogenesisof CNS injury, particularlyloss of dopaminergic neurons in the SN, in HIV-I encephalopathy, and on strategies for protecting from such neurotoxicity.
533. AAV-Mediated Aipl1 Gene Replacement in a Mouse Model of Leber Congenital Amaurosis
Claudio Mussolino,' Simona Neglia,' Chiara Abrescia,' Enrico M. Surace.I 'Telethon Institute ofGenetics and Medicine , TlGEM, Napoli, Italy.
Leber Congenital Amaurosis (LCA) is a severe retinal degenerative disease associated with mutations in genes preferentially expressed inphotoreceptors. LCAisgenetically heterogeneous and it Molecular Therapy Volume 15. Supplement 1. ~ b)' 2007 Copyright © The American Sex.."lety of Gene 'I11Cr:.lpy
S205
Thais Federici,' Robert H. Kutner.? Noel Tordo.' Jun Yang,' Erin Carlton,' Nicholas M. Boulis,' Jakob Reiser,' I Neurosciences, Cleve/and Clinic, Cleveland, OH; 2 Departm ent ofGene Therapy. Louisiana State University, Nell' Orleans, LA; "Lyssavirus Laboratory, Institut Pasteur; Paris, France .
Background: AAV and Rabies G-pseudotyped EIAV-based lentiviral vectors are the most promising vectors in the field of gene transfer and potential candidates for clinical applications for ncurodegenerative diseases. Lentiviral vectors derived from EIAV and HIV·I have twice the genomic capacity of AAV vectors, which allows insertion of additional transgenesequencesthat may improve their therapeutic outcome. The delivery of therapeutic proteins to the CNS representsan appealingstrategy for the treatmentof nerve injuryand disordersofthe CNS.lmportant factorsdeterminingCNS targeting includetropismand retrogradetransportof the vector particles. Herewe comparednovelpseudotyped HIV-I-basedlentiviral vectors, evaluating their ability to transduce specific cell populations and promote retrograde axonal transport in vitro. Methods: HIV-I-based vectors, pseudotyped with the G glycoproteins from dilTerent enveloped viruses belongingto the Rhabdoviridae family, genus Lyssavirus (RabiesG, European Bat, Lagos Bat, Duvenhage, VSV-G), were used. The vectors expressed the Enhanced Green Fluorescent Protein (EGFP) or the E. coli J3-galactosidase (LacZ) transgcncs, controlled by the constitutiveCytomegalovirus(CMVIE) promoter. Wetested their performancefor efficientdeliveryand expression of transgenes in NSC-34 cells and rat primary mixed spinal cord cultures. We then analyzed their uptake and retrograde transport in vitro, using Campenot chambers. We also constructed and tested VSV-G pseudotypedvectors,expressingthe EGFPtransgene and bearingdifferentpromotersincludingthe humanubiquitin C (hUbC) promoterand the human synapsin-I (hSYNI) promoter. Flowcytometry(FACS), X-galstaining,and immunocytochemistry were performed. Results: We had previously performed a in vitro study with SH-SY5Y cells and demonstrated that our Rabies Gpseudotyped HIV-I-based vector possessed the highest neuronal tropismamongthe pseudotypestested. In thisseries of experiments, the Rabies G-pseudotyped vector demonstrated again the highest levelsofJ3-Gal-positive cells in mixed spinal cord cultures,as well as the best uptake and retrogradetransport in Campenotchambers. Concerningthe promoters,all the vectors showed robust transgene expression in NSC-34 cells as judged by flow cytometry, The transgene expression with the hUbc and hSYNI promoters was strong and exclusivelyneuronal, whereas the transgeneexpression from the hCMV-IE promoter was robust, but also present in glial cells, as shown by immunocytochemistry. Conclusion: Our in vitro studies demonstrated that the Rabies G-pseudotyped vector possessed the best performance and neuronal tropism among the pseudotypes tested in vitro. Because the pattern of transduction in vitro docs not necessarily reflect the uptake in vivo, additional studiesarc underwayto confirmthe transductionpropertiesofthese vectors using an animal model. Acknowledgement : This research was supported by grants from NINDS K08 NS43305, ALSA and NINDS ROI NS44832.
532. Trans-Synaptic Neurotoxicity of HIV-1 gp120 and Trans-Synaptic Protection Using rSV40 Vectors Delivery Antioxidant Enzymes
Jean-Pierre Louboutin,' LokeshAgrawal; BeverlyA. S. Reyes,' ElisabethJ. van Bockstaele,' David S. Strayer,' I Pathology; Thomas Jefferson University, Philadelphia, PA; 'Neurosurgery and Farber Institute for Neurosciences, Thomas Jefferson University; Philadelphia, PA.
The basalgangliaare oftenaffectedin patientswith HIV-I-associated dementia (I·IAD). In addition to signs of neuron loss in many areas, Parkinson-like symptomatology is common in people with HAD.The neurotoxicityof HIV-I envelopeglycoprotein, gp120,is thoughtto be in partresponsible for the neuronlossin HADpatients. We developed an animal model to study gpl20-induced lesions in the rat brain by injectingvaryingdoses of gp120stereotaxically into the caudate-putamen (CP), then determining the extent of neuron loss. We then tested the ability of antioxidant gene transfer using recombinant SV40-derived vectors delivering CulZn superoxide dismutase (SV(SOD1» or glutathione peroxidase (SV(GPxl». Neuron loss was measured by TUNEL assay for apoptosis and by enumerating neurotrace-positive cells.Whenwe injected gp120(100 ng, 250 ng or 500 ng) into the CP, tissue damage extended several microns rostral and caudal to the injectionsite, as observed on neutral red (NR)-stainedcryostat sections 7 and 14days after injection. The area of the damage quantified by morphometric analysis on Nk-stained sections was about 25% of the total area of the Cp' 7 and 14days after injectionof500 nggpl20. (100 ng gpl20 induced minimal lesions). Staining of neurons by Neurotraee (NT) showed a loss of 30% of Nl-positivc cells during the same time period. Similar results were found on sections immunostained for neuN, a marker of neurons. However. neuronal loss was also observed in dilTerent cortical areas at 7 and 14 days after intra-CP injection of gp120. Specifically, in the striatum many neurons are involved in dopaminergic systemsand expressthe dopaminetransporter(OAT). Numbers of OAT-positive cells were decreased by 50% at 7 and 14 days after injectionof gp120 in the CPo A decrease in the number of tyrosine-hydroxylase (n-I)-positive neurons was also observed in the SN, the principal efTeetor of the dopaminergic system, from 4 to 14 days after injectionof gp120 in the CPo This finding suggests that gp120-induced neurotoxicity could extend beyond one and two synapses from the site of exposure. Although the mechanism by which this occurs is not clear, in another abstract, we document axonal transport of gp120. Furthermore, injection of SV(SOOI) or SV(GPxl) into the CP, from 4 to 24 weeks before injection of 500 ng gp120 in the Cp' significantlyand substantiallyreduced the extent of the injury, the loss of NT- and OAT-positive cells in the cr, as well as the diminution of the Tll-positive cells in the SN. Therefore, we demonstratethat the neurotoxicity ofl-IIV-I envelope gp120 can traverse synapses and that the protection afforded by prior gene transferof SODI or GPxI using rSV40 vectors may also traverse synapses.These findingsbear both on the pathogenesisof CNS injury, particularlyloss of dopaminergic neurons in the SN, in HIV-I encephalopathy, and on strategies for protecting from such neurotoxicity.
533. AAV-Mediated Aipl1 Gene Replacement in a Mouse Model of Leber Congenital Amaurosis
Claudio Mussolino,' Simona Neglia,' Chiara Abrescia,' Enrico M. Surace.I 'Telethon Institute ofGenetics and Medicine , TlGEM, Napoli, Italy.
Leber Congenital Amaurosis (LCA) is a severe retinal degenerative disease associated with mutations in genes preferentially expressed inphotoreceptors. LCAisgenetically heterogeneous and it Molecular Therapy Volume 15. Supplement 1. ~ b)' 2007 Copyright © The American Sex.."lety of Gene 'I11Cr:.lpy
S205