- Email: [email protected]
988. Antioxidant Enzymes Delivered Using SV40 Vectors Protect Primary Neurons In Vitro from Apoptosis Mediated by Transduced HIV-1 gp120
after nerve injury, rats treated with HSV-GDNF vector exhibited significant recovery of ICP/AP compared with control vector or untreated groups . The I-ISV-GDNF group also yielded more FGpositive MPG cells than the control vector group. Rats treated with HSV-neurturin exhibited significant recovery of ICP/AP compared with the HSV-LacZ group or HSV-untreated group at 4 weeks after the nerve injury. The HSV-neurturin group had more FG-positive cells in MPG than HSV-LacZ group . HSV vector-mediated delivery of GON r or neurturin presents a viable approach for treatment of erectile dysfunction (ED) following cavernous nerve injury.
987. Effects of Promoter and Viral Dose on AAV Vector-Mediated Gene Expression, Efficacy and Survival in a Mouse Model of Niemann-Pick A Disease
Jie Bu,1 James C. Dodge,' Catherine R. O'Riordan,' Robin J. Ziegler,' Antonius N. Song, I Joseph W. Foley,' Jonathan A. Fidler, l Eric M. Roskelley,' Edward 1-1. Schuchman.' Lamya S. Shihabuddin,' Seng H. Cheng ,' Marco A. Passini.' 'Neuroscience , Genzyme Corporation, Framingham, 1I1A; "HIIJIIan Genetics, Mount Sinai School ofMedicine, New York, NY.
Adeno-associated virus (AAV) vectors are efficient tools for delivering therapeutic genes to the CNS . Understanding the properties of recombinant AAV in the brain is a critical requisite to designing treatment strategies for neurological disorders. In this study, we utilized anAAV serotype-I vector that encoded for human acid sphingomyelinase (hASM) to determine long-term hASM expression from different promoters, and the relationship between AAV vector dose and therapeutic efficacy and survival in the ASM knock-out (ASMKO) mouse model ofNiemann-PickA disease. AAY1-hASM under the control ofeither the 0.7 kb cytomegalovirus (CMY), 1.6kb CMY enhancer-chicken p-actin (CBA), 0.5 kb synapsin (SYN) , 0.4 kb p-glucuronidase (GUSB), or 1.4 kb CMY enhanccr-human ubiquitin B (CUl3l) promoter injected into the brain ofC57/b6 mice and sacrificed at 1,4, and 24 months post-injection (pi). At I month pi, the CBAand CUBI promoters produced the highest levels ofhASM, which was significantly better than the SYN and CMV promoters. However, there was an eventual 30-50% decline ofexpression from all promoters by 4 months pi. Human ASM levels remained stable and did not show any further decline between 4 and 24 months pi. Similar, yet moderate levels of circulating anti-hASM antibodies were observed at 24 months pi in all promoter groups. The antibody response did not have long-term deleterious effects on expression as determined by the stable hASM levels, and by the robust hASM protein and mRNA staining on tissue sections at 24 months pi. We next evaluated the efficacy of different doses of recombinant AAY to reverse storage pathology and provide longevity benefits in the ASMKO mouse. Four doses ofAAV I-CBA-hASM that range from 1.2 ell genome copies (gc) to 4.0 e9 gc were injected into multiple regions of the mutant brain. As may be expected, a dose-response for hASM protein levels was exhibited in treated animals. However, all animals that received virus in the log range of 1.2 e II to 1.2 e lOge showed a similar and significant decrease in sphingomyelin storage in the brain . All doses in the log range of 4.0 e 10 to 4.0 e9 gc showed prolongedsurvival with a median life-span of 42 weeks compared to 34 weeks in untreated ASMKO mice. Mice receiving the highest dose, 1.2 e l l ge, showed a superior increase in survival with a median life span of52 weeks . Thus , while it may be possible to decrease storage burden with lower doses of AAY vectors , the most efficacious survival benefit inASMKO mice was only realized with the highest dose.
Molecular Therapy \ '01"", < 15. Suppl ement I•.\1,,)"2007 O:lprnghf © TIl[: American $Ol;:it,:'t}, or (if,.'ru,; Therapy
988. Antioxidant Enzymes Delivered Using SV40 Vectors Protect Primary Neurons In Vitro from Apoptosis Mediated by Transduced HIV-1 gp120
Lokesh Agrawal,' J.-P. Louboutin,' Beverly A. S. Reyes,' Elisabeth Van Bockstaele.? David S. Strayer. I 'Pathology, Thomas Jefferson University. Philadelphia. I'll; Wellrosurgery, Thomas Jefferson University. Philadelphia, PII.
HIY-I infection in brain is associated with cognitive impairment and with neuronal apoptosis in the hippocampus, basal ganglia and cerebral cortex. Neuron loss leads to mild motor and sensory neurological deficits to severe dementia HIV-I-associated dementia (HAD) involve neuron damage mediated by J-[IV-l gene products which cause oxidation of neuronal proteins and cytotoxic levels of lipid peroxidation. Among the key I-IIV-I gene products in J-[IY encephalopathy is the envelope (Env) glycoprotein, gp 120.1nAIDS patients, CNS damage is not acute, but rather slowly progressive, as continued production of HIY-I proteins by infected microglia and macrophages in the brain leads to neuron loss. An accessible model of the type of chronic cell injury seen in I-IIY-l-induced encephalopathy has not been yet entirely established. We have created a model for gp 120-induced apoptosis that depends on continued expression of one of the neurotoxic HIV-I gene products, gpl20 using a Tag-deleted recombinant SY40 vector to transduce primary neurons and provide continued gpl20 expression. We studied the parameters ofthat system with regard to expression of gp 120, neuron apoptosis, protein oxidation and lipid peroxidation. We compared neuron cultures transduced with SY(gp 120) with those exposed to recombinant gp 120 alone, and studied the effectiveness of'therapeutic intervention in vitro using rSV40-delivered antioxidant enzymes. SV(gp 120) induced significant apoptosis in primary neurons, compared to mock-transduced cultures or cultures treated with a control rSY40 vector. Lipid peroxidation was studied using a calorimetric malonaldehyde (MDA) assay. We found that neurons transduced with SY(gpI20) showed 2-3 fold greater MDA(3-4 mM) levels, compared with control cultures . Primary neurons were first treated with SY(SOD I) or SY(GPxl) and then challenged with SV(gp 120). The levels ofMDA and protein oxidation were also quantitated later. We studied these measurements using calorimetric rnalonaldehydc (MDA) assays and using oxyblot protein oxidation assays. These findings are parallel to observations following acute exposure to recom binant gp 120. Transduction of cultured neurons with rSV40 vectors bearing Cu/Zn superoxide dismutase (SY(SODI» or glutathione peroxidase (SY(GPxl» substantially protected them from apoptosis caused by transduction with SY(gp 120). Intersetingly, transduction with SY(GPxl), but not SV(SODI), led to significantly less MDA release as compared to controls after challenge with SY(gpI20). Treating neurons with SY(gpI20) also resulted in oxidation of several proteins with prominent bands at 40 kDa and 25 kDa. Currently we are characterizing these proteins. We are also in the process of determining the effectiveness of rSY40delivered antioxidant enzymes in preventing the protein oxidation due to SY(gpI20) treatment. Understanding the role of chronic production of gp 120 in mediating apoptosis, protein oxidation and lipid pcroxidation with subsequent protection using SY(SOD I) or SY(GPx I) may help develop new therapeutic strategies for dementia associated with HIY.
S377
987. Effects of Promoter and Viral Dose on AAV Vector-Mediated Gene Expression, Efficacy and Survival in a Mouse Model of Niemann-Pick A Disease
Jie Bu,1 James C. Dodge,' Catherine R. O'Riordan,' Robin J. Ziegler,' Antonius N. Song, I Joseph W. Foley,' Jonathan A. Fidler, l Eric M. Roskelley,' Edward 1-1. Schuchman.' Lamya S. Shihabuddin,' Seng H. Cheng ,' Marco A. Passini.' 'Neuroscience , Genzyme Corporation, Framingham, 1I1A; "HIIJIIan Genetics, Mount Sinai School ofMedicine, New York, NY.
Adeno-associated virus (AAV) vectors are efficient tools for delivering therapeutic genes to the CNS . Understanding the properties of recombinant AAV in the brain is a critical requisite to designing treatment strategies for neurological disorders. In this study, we utilized anAAV serotype-I vector that encoded for human acid sphingomyelinase (hASM) to determine long-term hASM expression from different promoters, and the relationship between AAV vector dose and therapeutic efficacy and survival in the ASM knock-out (ASMKO) mouse model ofNiemann-PickA disease. AAY1-hASM under the control ofeither the 0.7 kb cytomegalovirus (CMY), 1.6kb CMY enhancer-chicken p-actin (CBA), 0.5 kb synapsin (SYN) , 0.4 kb p-glucuronidase (GUSB), or 1.4 kb CMY enhanccr-human ubiquitin B (CUl3l) promoter injected into the brain ofC57/b6 mice and sacrificed at 1,4, and 24 months post-injection (pi). At I month pi, the CBAand CUBI promoters produced the highest levels ofhASM, which was significantly better than the SYN and CMV promoters. However, there was an eventual 30-50% decline ofexpression from all promoters by 4 months pi. Human ASM levels remained stable and did not show any further decline between 4 and 24 months pi. Similar, yet moderate levels of circulating anti-hASM antibodies were observed at 24 months pi in all promoter groups. The antibody response did not have long-term deleterious effects on expression as determined by the stable hASM levels, and by the robust hASM protein and mRNA staining on tissue sections at 24 months pi. We next evaluated the efficacy of different doses of recombinant AAY to reverse storage pathology and provide longevity benefits in the ASMKO mouse. Four doses ofAAV I-CBA-hASM that range from 1.2 ell genome copies (gc) to 4.0 e9 gc were injected into multiple regions of the mutant brain. As may be expected, a dose-response for hASM protein levels was exhibited in treated animals. However, all animals that received virus in the log range of 1.2 e II to 1.2 e lOge showed a similar and significant decrease in sphingomyelin storage in the brain . All doses in the log range of 4.0 e 10 to 4.0 e9 gc showed prolongedsurvival with a median life-span of 42 weeks compared to 34 weeks in untreated ASMKO mice. Mice receiving the highest dose, 1.2 e l l ge, showed a superior increase in survival with a median life span of52 weeks . Thus , while it may be possible to decrease storage burden with lower doses of AAY vectors , the most efficacious survival benefit inASMKO mice was only realized with the highest dose.
Molecular Therapy \ '01"", < 15. Suppl ement I•.\1,,)"2007 O:lprnghf © TIl[: American $Ol;:it,:'t}, or (if,.'ru,; Therapy
988. Antioxidant Enzymes Delivered Using SV40 Vectors Protect Primary Neurons In Vitro from Apoptosis Mediated by Transduced HIV-1 gp120
Lokesh Agrawal,' J.-P. Louboutin,' Beverly A. S. Reyes,' Elisabeth Van Bockstaele.? David S. Strayer. I 'Pathology, Thomas Jefferson University. Philadelphia. I'll; Wellrosurgery, Thomas Jefferson University. Philadelphia, PII.
HIY-I infection in brain is associated with cognitive impairment and with neuronal apoptosis in the hippocampus, basal ganglia and cerebral cortex. Neuron loss leads to mild motor and sensory neurological deficits to severe dementia HIV-I-associated dementia (HAD) involve neuron damage mediated by J-[IV-l gene products which cause oxidation of neuronal proteins and cytotoxic levels of lipid peroxidation. Among the key I-IIV-I gene products in J-[IY encephalopathy is the envelope (Env) glycoprotein, gp 120.1nAIDS patients, CNS damage is not acute, but rather slowly progressive, as continued production of HIY-I proteins by infected microglia and macrophages in the brain leads to neuron loss. An accessible model of the type of chronic cell injury seen in I-IIY-l-induced encephalopathy has not been yet entirely established. We have created a model for gp 120-induced apoptosis that depends on continued expression of one of the neurotoxic HIV-I gene products, gpl20 using a Tag-deleted recombinant SY40 vector to transduce primary neurons and provide continued gpl20 expression. We studied the parameters ofthat system with regard to expression of gp 120, neuron apoptosis, protein oxidation and lipid peroxidation. We compared neuron cultures transduced with SY(gp 120) with those exposed to recombinant gp 120 alone, and studied the effectiveness of'therapeutic intervention in vitro using rSV40-delivered antioxidant enzymes. SV(gp 120) induced significant apoptosis in primary neurons, compared to mock-transduced cultures or cultures treated with a control rSY40 vector. Lipid peroxidation was studied using a calorimetric malonaldehyde (MDA) assay. We found that neurons transduced with SY(gpI20) showed 2-3 fold greater MDA(3-4 mM) levels, compared with control cultures . Primary neurons were first treated with SY(SOD I) or SY(GPxl) and then challenged with SV(gp 120). The levels ofMDA and protein oxidation were also quantitated later. We studied these measurements using calorimetric rnalonaldehydc (MDA) assays and using oxyblot protein oxidation assays. These findings are parallel to observations following acute exposure to recom binant gp 120. Transduction of cultured neurons with rSV40 vectors bearing Cu/Zn superoxide dismutase (SY(SODI» or glutathione peroxidase (SY(GPxl» substantially protected them from apoptosis caused by transduction with SY(gp 120). Intersetingly, transduction with SY(GPxl), but not SV(SODI), led to significantly less MDA release as compared to controls after challenge with SY(gpI20). Treating neurons with SY(gpI20) also resulted in oxidation of several proteins with prominent bands at 40 kDa and 25 kDa. Currently we are characterizing these proteins. We are also in the process of determining the effectiveness of rSY40delivered antioxidant enzymes in preventing the protein oxidation due to SY(gpI20) treatment. Understanding the role of chronic production of gp 120 in mediating apoptosis, protein oxidation and lipid pcroxidation with subsequent protection using SY(SOD I) or SY(GPx I) may help develop new therapeutic strategies for dementia associated with HIY.
S377