- Email: [email protected]
431. In Utero CNS Delivery of an Integrating HSV-1 Amplicon Vector Leads to Prolonged, Neuron-Specific Gene Expression
NEUROLOGIC DISORDERS specifically the hARSA, immunohistochemistry and measurement of hARSA activity with the artificial substrate p-nitrocatechol sulfate. As expected, hARSA was mostly expressed in neurons, but also in a significant percentage of astrocytes (up to 14% arround the injection site) and in microglial cells. The widespread diffusion of hARSA resulted from both vector and enzyme diffusion within the CNS. Quantitative PCR in AAV5-ARSA vector showed that vector genomes were concentrated at the injection sites but spreads over a distance from injection sites. ARSA expression using ELISA test was proportional to the number of vector genomes but extended far beyond PCR-positive brain regions. The increase of ARSA positive neurons and astrocytes from 6 to 18 months of age confirmed that a significant amount of AAV5-encoded ARSA was secreted by transduced cells and captured by neurons and astrocytes. ARSA delivery prevented sulfatide deposits and reversed the galactosyl ceramide/sulfatide abnormal ratio that reflects sulfatide storage and loss of oligodendrocytes. Eighteen-month old treated mice showed a nearly absence of neuropathological abnormalities (Purkinje cell loss, astrogliosis, microglial activation) and normal motor behavior (at the rotarod test). These results indicate that ARSA delivery from intracerebral sources has the capacity to alleviate most disease manifestation in the MLD model.
430. Immune System Regulation of Transgene Expression from High-Capacity Gutless Adenoviral Vectors in the Mouse Brain: Very Long Term Expression Following Pre-Immunization Carlos Barcia,1 Jeffrey M. Zirger,1 Weidong Xiong,1 Philip Ng,3 Donna Palmer,3 Maria G. Castro,1,2 Pedro R. Lowenstein.1,2 1 Gene Therapeutics Research Institute, Cedars-Sinai Medical Center; 2Molecular and Medical Pharmacology, School of Medicine, University of California at Los Angeles, Los Angeles, CA; 3Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX. Gene expression from first generation adenovirus vectors in the CNS is efficient and safe in naïve animals. However, transgene expression is eliminated within 30 days in animals immunized against adenovirus. High-capacity adenovirus (HC-Adv) provide an advantage in that they sustain transgene expression in the brain even in animals that have been preimmunized against adenovirus, preceding the administration of HC-Adv. In the present experiment we analyzed the long term expression of a HC-Adv in the brain of mice preimmunized with a first generation adenovirus. C57Bl/6 mice were immunized ip with 1x108 i.u. of RAdHPRT, and 30 days later they were injected in the striatum with 1x106 bfu of HC-Adv-STkmCMVLacZ. Mice were perfused-fixed at different time points after the intrastriatal injection (14, 30, 60 90 and 180 days). Brains were then removed and cut in 40mm thick serial sections. Immunohistochemistry for β-galactosidade was performed and expressing cells and fibers were counted by quantitative unbiased stereology. After 14 days the expression in the striatum was high covering the entire caudate-putamen complex. Fibers and neurons were observed in the motor cortex, globus pallidus, entopeduncular nucleus, amygdaloid complex and Substantia nigra pars compacta. 30 days after the injection, the expression of the transgene in the striatum was diminished 30% in non immunized animals, while in immunized animals it was reduced by more than 50%. However after 60 days the expression was stable in both groups, and this expression lasted for up to 6 months. These results confirm the stable expression of the transgene in the brain with HC-Adv even in animals immunized systemically before the delivery of HC-Adv into the brain, and demonstrate the usefulness of HC-Adv as powerful tool for gene therapy for the treatment of neurological disorders such as Parkinson’s and Huntington’s disease. Molecular Therapy Volume 11, Supplement 1, May 2005 Copyright The American Society of Gene Therapy
431. In Utero CNS Delivery of an Integrating HSV-1 Amplicon Vector Leads to Prolonged, Neuron-Specific Gene Expression William J. Bowers,1,2 Michael A. Mastrangelo,2 Ann E. Casey,2 Hilary A. Southerland,2 Kathleen A. Maguire-Zeiss,1,2 Howard J. Federoff.1,2 1 Neurology, Univ. of Rochester Sch. of Med. and Dent., Rochester, NY; 2Ctr. for Aging and Dev. Biol., Univ. of Rochester Sch. of Med. and Dent., Rochester, NY; 3Rochester, NY. Correction of brain diseases diagnosed prenatally could be achieved if an efficient and safe in utero gene transfer vehicle were available. The HSV-1-derived amplicon, a vector devoid of viral genes and with the largest payload capacity, normally exists episomally within transduced cells. Persistent expression in dividing cells has proven elusive since the amplicon is replication-defective. Developing an integrating form of the HSV amplicon would undoubtedly enhance the versatility of this vector platform. Here we describe combining the Tc1-like Sleeping Beauty (SB) transposon system with the amplicon to engineer a vector that possesses the ability to provide brain-wide and prolonged transgene expression in neurons following in utero vector infusion. Two vectors were constructed: one containing an SV40 promoter-driven β-galactosidase-neomycin (βgeo) fusion transgene flanked by the SB inverted/direct repeats (HSVT-βgeo), and a second containing the SB transposase gene transcriptionally driven by the HSV immediate-early 4/5 gene promoter (HSVsb). Co-delivery of these vectors to the brains of E14.5 C57BL/6 mouse embryos resulted in the birth of viable neonates, integration of the transposable element from HSVT-βgeo, and extended transgene expression duration (at least 90 days) when compared to embryos transduced with HSVT-βgeo and empty vector control, HSVPrPUC. With its uniquely large payload capacity this new amplicon platform enables novel treatments of pre- and postnatal brain diseases. Consultant for AmpliVex, LLC.
432. Molecular Chimerism Prevents Experimental Autoimmune Encephalomyelitis (EAE) Herena Eixarch,1 Carmen Espejo,2 Francisco Vidal,1 Silvia Garces,2 Mireia Castillo,2 Alex Bote,3 Elisabeth Kadar,1 Marta Rosal,3 Xavier Montalban,2 Jordi Barquinero.1 1 Unitat de Diagnostic i Terapia Molecular, Centre de Transfusio i Banc de Teixits, Barcelona, Spain; 2Unitat de Neuroimmunologia Clinica, Hospital Universitari Vall Hebron, Barcelona, Spain; 3 Unitat Cientificotecnica de Suport, Institut Fundacio de Recerca Hospital Universitari Vall Hebron, Barcelona, Spain. Molecular chimerism in the hematopoietic system is usually associated with immune tolerance to the transgene products. Experimental autoimmune encephalomyelitis (EAE), a model of multiple sclerosis (MS), is a CD4+T cell mediated inflammatory and demyelinating disease that can be induced in susceptible animals upon immunization with certain proteins or peptides present in the myelin sheet, which results in an ascending paralysis. We hypothesized that stably expressing the encephalitogenic peptide containing 16 aminoacids of the myelin oligodendrocyte glycoprotein (MOG40-55) in the murine marrow cells will induce specific immune tolerance to the antigen, which will prevent or reduce susceptibility to the disease. To this end, we constructed a retroviral vector containing the coding sequence of the invariant chain (CD74), in which the CLIP region was replaced by a sequence encoding the MOG40-55 peptide, in order to target its expression to the MHC class II compartment. EGFP was also included in the vector as a reporter gene. Groups of mice (n = 4 and 8, respectively, in two separate experiments) were conditioned with non-myeloablative S167
430. Immune System Regulation of Transgene Expression from High-Capacity Gutless Adenoviral Vectors in the Mouse Brain: Very Long Term Expression Following Pre-Immunization Carlos Barcia,1 Jeffrey M. Zirger,1 Weidong Xiong,1 Philip Ng,3 Donna Palmer,3 Maria G. Castro,1,2 Pedro R. Lowenstein.1,2 1 Gene Therapeutics Research Institute, Cedars-Sinai Medical Center; 2Molecular and Medical Pharmacology, School of Medicine, University of California at Los Angeles, Los Angeles, CA; 3Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX. Gene expression from first generation adenovirus vectors in the CNS is efficient and safe in naïve animals. However, transgene expression is eliminated within 30 days in animals immunized against adenovirus. High-capacity adenovirus (HC-Adv) provide an advantage in that they sustain transgene expression in the brain even in animals that have been preimmunized against adenovirus, preceding the administration of HC-Adv. In the present experiment we analyzed the long term expression of a HC-Adv in the brain of mice preimmunized with a first generation adenovirus. C57Bl/6 mice were immunized ip with 1x108 i.u. of RAdHPRT, and 30 days later they were injected in the striatum with 1x106 bfu of HC-Adv-STkmCMVLacZ. Mice were perfused-fixed at different time points after the intrastriatal injection (14, 30, 60 90 and 180 days). Brains were then removed and cut in 40mm thick serial sections. Immunohistochemistry for β-galactosidade was performed and expressing cells and fibers were counted by quantitative unbiased stereology. After 14 days the expression in the striatum was high covering the entire caudate-putamen complex. Fibers and neurons were observed in the motor cortex, globus pallidus, entopeduncular nucleus, amygdaloid complex and Substantia nigra pars compacta. 30 days after the injection, the expression of the transgene in the striatum was diminished 30% in non immunized animals, while in immunized animals it was reduced by more than 50%. However after 60 days the expression was stable in both groups, and this expression lasted for up to 6 months. These results confirm the stable expression of the transgene in the brain with HC-Adv even in animals immunized systemically before the delivery of HC-Adv into the brain, and demonstrate the usefulness of HC-Adv as powerful tool for gene therapy for the treatment of neurological disorders such as Parkinson’s and Huntington’s disease. Molecular Therapy Volume 11, Supplement 1, May 2005 Copyright The American Society of Gene Therapy
431. In Utero CNS Delivery of an Integrating HSV-1 Amplicon Vector Leads to Prolonged, Neuron-Specific Gene Expression William J. Bowers,1,2 Michael A. Mastrangelo,2 Ann E. Casey,2 Hilary A. Southerland,2 Kathleen A. Maguire-Zeiss,1,2 Howard J. Federoff.1,2 1 Neurology, Univ. of Rochester Sch. of Med. and Dent., Rochester, NY; 2Ctr. for Aging and Dev. Biol., Univ. of Rochester Sch. of Med. and Dent., Rochester, NY; 3Rochester, NY. Correction of brain diseases diagnosed prenatally could be achieved if an efficient and safe in utero gene transfer vehicle were available. The HSV-1-derived amplicon, a vector devoid of viral genes and with the largest payload capacity, normally exists episomally within transduced cells. Persistent expression in dividing cells has proven elusive since the amplicon is replication-defective. Developing an integrating form of the HSV amplicon would undoubtedly enhance the versatility of this vector platform. Here we describe combining the Tc1-like Sleeping Beauty (SB) transposon system with the amplicon to engineer a vector that possesses the ability to provide brain-wide and prolonged transgene expression in neurons following in utero vector infusion. Two vectors were constructed: one containing an SV40 promoter-driven β-galactosidase-neomycin (βgeo) fusion transgene flanked by the SB inverted/direct repeats (HSVT-βgeo), and a second containing the SB transposase gene transcriptionally driven by the HSV immediate-early 4/5 gene promoter (HSVsb). Co-delivery of these vectors to the brains of E14.5 C57BL/6 mouse embryos resulted in the birth of viable neonates, integration of the transposable element from HSVT-βgeo, and extended transgene expression duration (at least 90 days) when compared to embryos transduced with HSVT-βgeo and empty vector control, HSVPrPUC. With its uniquely large payload capacity this new amplicon platform enables novel treatments of pre- and postnatal brain diseases. Consultant for AmpliVex, LLC.
432. Molecular Chimerism Prevents Experimental Autoimmune Encephalomyelitis (EAE) Herena Eixarch,1 Carmen Espejo,2 Francisco Vidal,1 Silvia Garces,2 Mireia Castillo,2 Alex Bote,3 Elisabeth Kadar,1 Marta Rosal,3 Xavier Montalban,2 Jordi Barquinero.1 1 Unitat de Diagnostic i Terapia Molecular, Centre de Transfusio i Banc de Teixits, Barcelona, Spain; 2Unitat de Neuroimmunologia Clinica, Hospital Universitari Vall Hebron, Barcelona, Spain; 3 Unitat Cientificotecnica de Suport, Institut Fundacio de Recerca Hospital Universitari Vall Hebron, Barcelona, Spain. Molecular chimerism in the hematopoietic system is usually associated with immune tolerance to the transgene products. Experimental autoimmune encephalomyelitis (EAE), a model of multiple sclerosis (MS), is a CD4+T cell mediated inflammatory and demyelinating disease that can be induced in susceptible animals upon immunization with certain proteins or peptides present in the myelin sheet, which results in an ascending paralysis. We hypothesized that stably expressing the encephalitogenic peptide containing 16 aminoacids of the myelin oligodendrocyte glycoprotein (MOG40-55) in the murine marrow cells will induce specific immune tolerance to the antigen, which will prevent or reduce susceptibility to the disease. To this end, we constructed a retroviral vector containing the coding sequence of the invariant chain (CD74), in which the CLIP region was replaced by a sequence encoding the MOG40-55 peptide, in order to target its expression to the MHC class II compartment. EGFP was also included in the vector as a reporter gene. Groups of mice (n = 4 and 8, respectively, in two separate experiments) were conditioned with non-myeloablative S167