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350. Kidney-Directed Gene Therapy for Murine Glycogen Storage Disease Type IA
Diabetes, Metabolic and Genetic Diseases II 349. The Cure of Canavan Disease: Is It a Scientific Fiction or Clinical Reality?
Dominic J. Gessler, Danning Li, Hongxia Xu, Qin Su, Guangping Gao Gene Therapy Center, UMass, Worcester, MA Canavan Disease (CD) is a rare and lethal inherited pediatric CNS disorder with recessive mutations in the aspartoacylase (ASPA) gene. Traditionally, its variable disease phenotypes were descripted with the congenital sub-form showing neonatal onset and the severest phenotype with early death. The two other sub-forms, infantile, and juvenile, manifest with delayed onset and milder symptoms. Some CD patients are now 20 years of age and older. To date, there is no effective treatment available. Thus, gene replacement therapy is an attractive approach for treating this devastating disease. Previously, we have shown that a single intravenous (i.v.) injection of recombinant adenoassociated virus (rAAV) expressing human ASPA (hASPA) rescues early lethality and partially restores motor function (1st generation gene therapy) in the CD knock-out (CD KO) mouse, which resembles the congenital sub-form of CD and displays the severest phenotype of all available CD mouse models, with early death at around post-natal day (p) 28. After this remarkable improvement of symptoms with our 1st generation treatment, we further optimized our gene therapy. Now, in its 2nd and 3rd generation, our gene therapy cures the disease in the CD KO mouse. Interestingly, our 3rd generation gene therapy turns CD KO mice into “supermice”, outperforming WT mice on rotarod motor function test. This rescue is persistent and currently mice at 1.5 years of age still show no signs of disease reoccurrence. CNS pathology and magnet resonance imaging (MRI) at p25 and p365 show complete normalization. To further support the efficacy of our 3rd generation gene therapy, we performed neurometabolome profiling with over 400 characterized metabolites that showed reversal of the Canavan disease related metabolic changes including myelin associated lipids. To further evaluate the potency of our 3rd generation gene therapy, we tested different doses and routes of administration. Of note, 200-fold lower doses intraventricularly (ICV) administered still rescues lethality, while mice treated ICV with 20-fold reduced dose draw even with WT mice on motor function testing. Next, we moved to the Nur7 mouse model (resembles infantile and juvenile sub-form) that displays a similar disease pattern as the CD KO mouse with respect to growth curve and neurologic symptoms but eventually re-gains weight and shows survival similar to wild-type mice. Again, we treated mice i.v. with a single dose of rAAVhASPA at p1 as our gold standard and subsequent groups at 6 and 12 weeks of age to determine the therapeutic window. Of note, mice treated at 6 weeks of age recovered within 4 weeks post-treatment. Mice treated later than 6 weeks require more time to recover but still showed significant improvement over Nur7 mutants. This recovery was also correlated by CNS pathology and MRI. Currently, we are evaluating mice that were treated at 24 weeks of age to determine if there is a time point of no return. Overall, our data show clear evidence for the cure of the disease at early and late stages of the disease in two different mouse models. In addition, this is confirmed on different levels of cellular complexity by MRI, fMRI, CNS pathology, and neurometabolic profiling.
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350. Kidney-Directed Gene Therapy for Murine Glycogen Storage Disease Type IA
Youngmok Lee1, Seongho Bae2, Celine J. Rocca3, Stephanie Cherqui3, David A. Weinstein1, Janice Y. Chou2 1 Glycogen Storage Disease Program, Division of Pediatric Endocrinology, Department of Pediatrics, University of Florida, Gainesville, FL, 2Section on Cellular Differentiation, Division on Translational Medidine, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD, 3Department of Pediatrics, University of California, San Diego, La Jolla, CA Glycogen storage disease type Ia (GSD-Ia, MIM232200) is an autosomal recessive disorder caused by deficiencies in glucose-6phosphatase-α (G6Pase-α or G6PC) that is expressed primarily in the liver and kidney. GSD-Ia patients manifest impaired glucose homeostasis and a long-term complication of renal disease and there is no existing therapy to address this complication. We have previously shown that systemic administration of rAAV8-G6PC, a rAAV8 vector expressing human G6Pase-α directed by the human G6PC promoter/enhancer, delivers the G6Pase-α transgene to the liver of G6Pase-α-deficient (G6pc-/-) mice and corrects hepatic G6Pase-α deficiency. However, the rAAV8-G6PC vector transduces the kidney poorly and the treated G6pc-/- mice continued manifest renal dysfunction. In this study, we used the rAAV9-G6PC vector in a kidney-targeted gene delivery to improve renal function. The G6pc-/- die early even with glucose therapy. To overcome this, we performed a two-step kidney-directed gene delivery, first neonatally via the temporal vein with rAAV8-G6PC to sustain the survival of the mice, then at 12 weeks of age via retrograde renal vein injection with rAAV9-G6PC. Metabolic profiles and renal function were examined over a 52-week study. The rAAV-treated G6pc-/- mice exhibited normal fasting glucose profiles and could sustain a 24 hour of fast. Moreover, the treated G6pc-/- mice displayed normalized blood urea nitrogen concentrations, indicative of improved renal function. Our results strongly suggest that kidney-directed gene delivery with the rAAV9-G6PC vectors offers a promising and efficacious treatment for renal disease in GSD-Ia.
351. Efficacious Non-Oligodendrocyte Gene Therapy Suggests a New Dogma About CNS Compartmentalization of NAA Metabolism and Supports a Metabolic Sink Theory
Dominic J. Gessler1, Danning Li1, Hongxia Xu1, Qin Su1, Reuben Matalon2, Guangping Gao1 1 Gene Therapy Center, UMass, Worcester, MA, 2University of Texas Medical Branch, Galveston, TX N-acetylaspartate (NAA) is the second most abundant amino acid derivative in the mammalian central nervous system (CNS). Although its physiologic function remains elusive, many CNS disorders have been associated with changes in NAA levels, e.g. Alzheimer’s disease, bipolar disorder. The disease that is known to have direct connection to altered NAA metabolism is Canavan disease (CD); a leukodystrophy caused by mutations in the aspartoacylase (ASPA) gene. The current understanding is that, physiologically, the ASPA enzyme hydrolyzes NAA into L-aspartate and acetate in oligodendrocytes. Consequently, it was postulated that Canavan gene therapy has to restore ASPA expression in oligodendrocytes. However, we hypothesized that NAA can move freely and its cell-type independent break-down ameliorates Canavan disease. We constructed several tissue/cellspecific expression cassettes limiting hASPA expression to either astrocytes, neurons, oligodendrocytes, liver, heart, or muscle. We opted for the Canavan disease knock-out (CD KO) mouse model because it shows early lethality at around post-natal day 28 and the Molecular Therapy Volume 24, Supplement 1, May 2016 Copyright © The American Society of Gene & Cell Therapy
Dominic J. Gessler, Danning Li, Hongxia Xu, Qin Su, Guangping Gao Gene Therapy Center, UMass, Worcester, MA Canavan Disease (CD) is a rare and lethal inherited pediatric CNS disorder with recessive mutations in the aspartoacylase (ASPA) gene. Traditionally, its variable disease phenotypes were descripted with the congenital sub-form showing neonatal onset and the severest phenotype with early death. The two other sub-forms, infantile, and juvenile, manifest with delayed onset and milder symptoms. Some CD patients are now 20 years of age and older. To date, there is no effective treatment available. Thus, gene replacement therapy is an attractive approach for treating this devastating disease. Previously, we have shown that a single intravenous (i.v.) injection of recombinant adenoassociated virus (rAAV) expressing human ASPA (hASPA) rescues early lethality and partially restores motor function (1st generation gene therapy) in the CD knock-out (CD KO) mouse, which resembles the congenital sub-form of CD and displays the severest phenotype of all available CD mouse models, with early death at around post-natal day (p) 28. After this remarkable improvement of symptoms with our 1st generation treatment, we further optimized our gene therapy. Now, in its 2nd and 3rd generation, our gene therapy cures the disease in the CD KO mouse. Interestingly, our 3rd generation gene therapy turns CD KO mice into “supermice”, outperforming WT mice on rotarod motor function test. This rescue is persistent and currently mice at 1.5 years of age still show no signs of disease reoccurrence. CNS pathology and magnet resonance imaging (MRI) at p25 and p365 show complete normalization. To further support the efficacy of our 3rd generation gene therapy, we performed neurometabolome profiling with over 400 characterized metabolites that showed reversal of the Canavan disease related metabolic changes including myelin associated lipids. To further evaluate the potency of our 3rd generation gene therapy, we tested different doses and routes of administration. Of note, 200-fold lower doses intraventricularly (ICV) administered still rescues lethality, while mice treated ICV with 20-fold reduced dose draw even with WT mice on motor function testing. Next, we moved to the Nur7 mouse model (resembles infantile and juvenile sub-form) that displays a similar disease pattern as the CD KO mouse with respect to growth curve and neurologic symptoms but eventually re-gains weight and shows survival similar to wild-type mice. Again, we treated mice i.v. with a single dose of rAAVhASPA at p1 as our gold standard and subsequent groups at 6 and 12 weeks of age to determine the therapeutic window. Of note, mice treated at 6 weeks of age recovered within 4 weeks post-treatment. Mice treated later than 6 weeks require more time to recover but still showed significant improvement over Nur7 mutants. This recovery was also correlated by CNS pathology and MRI. Currently, we are evaluating mice that were treated at 24 weeks of age to determine if there is a time point of no return. Overall, our data show clear evidence for the cure of the disease at early and late stages of the disease in two different mouse models. In addition, this is confirmed on different levels of cellular complexity by MRI, fMRI, CNS pathology, and neurometabolic profiling.
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350. Kidney-Directed Gene Therapy for Murine Glycogen Storage Disease Type IA
Youngmok Lee1, Seongho Bae2, Celine J. Rocca3, Stephanie Cherqui3, David A. Weinstein1, Janice Y. Chou2 1 Glycogen Storage Disease Program, Division of Pediatric Endocrinology, Department of Pediatrics, University of Florida, Gainesville, FL, 2Section on Cellular Differentiation, Division on Translational Medidine, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD, 3Department of Pediatrics, University of California, San Diego, La Jolla, CA Glycogen storage disease type Ia (GSD-Ia, MIM232200) is an autosomal recessive disorder caused by deficiencies in glucose-6phosphatase-α (G6Pase-α or G6PC) that is expressed primarily in the liver and kidney. GSD-Ia patients manifest impaired glucose homeostasis and a long-term complication of renal disease and there is no existing therapy to address this complication. We have previously shown that systemic administration of rAAV8-G6PC, a rAAV8 vector expressing human G6Pase-α directed by the human G6PC promoter/enhancer, delivers the G6Pase-α transgene to the liver of G6Pase-α-deficient (G6pc-/-) mice and corrects hepatic G6Pase-α deficiency. However, the rAAV8-G6PC vector transduces the kidney poorly and the treated G6pc-/- mice continued manifest renal dysfunction. In this study, we used the rAAV9-G6PC vector in a kidney-targeted gene delivery to improve renal function. The G6pc-/- die early even with glucose therapy. To overcome this, we performed a two-step kidney-directed gene delivery, first neonatally via the temporal vein with rAAV8-G6PC to sustain the survival of the mice, then at 12 weeks of age via retrograde renal vein injection with rAAV9-G6PC. Metabolic profiles and renal function were examined over a 52-week study. The rAAV-treated G6pc-/- mice exhibited normal fasting glucose profiles and could sustain a 24 hour of fast. Moreover, the treated G6pc-/- mice displayed normalized blood urea nitrogen concentrations, indicative of improved renal function. Our results strongly suggest that kidney-directed gene delivery with the rAAV9-G6PC vectors offers a promising and efficacious treatment for renal disease in GSD-Ia.
351. Efficacious Non-Oligodendrocyte Gene Therapy Suggests a New Dogma About CNS Compartmentalization of NAA Metabolism and Supports a Metabolic Sink Theory
Dominic J. Gessler1, Danning Li1, Hongxia Xu1, Qin Su1, Reuben Matalon2, Guangping Gao1 1 Gene Therapy Center, UMass, Worcester, MA, 2University of Texas Medical Branch, Galveston, TX N-acetylaspartate (NAA) is the second most abundant amino acid derivative in the mammalian central nervous system (CNS). Although its physiologic function remains elusive, many CNS disorders have been associated with changes in NAA levels, e.g. Alzheimer’s disease, bipolar disorder. The disease that is known to have direct connection to altered NAA metabolism is Canavan disease (CD); a leukodystrophy caused by mutations in the aspartoacylase (ASPA) gene. The current understanding is that, physiologically, the ASPA enzyme hydrolyzes NAA into L-aspartate and acetate in oligodendrocytes. Consequently, it was postulated that Canavan gene therapy has to restore ASPA expression in oligodendrocytes. However, we hypothesized that NAA can move freely and its cell-type independent break-down ameliorates Canavan disease. We constructed several tissue/cellspecific expression cassettes limiting hASPA expression to either astrocytes, neurons, oligodendrocytes, liver, heart, or muscle. We opted for the Canavan disease knock-out (CD KO) mouse model because it shows early lethality at around post-natal day 28 and the Molecular Therapy Volume 24, Supplement 1, May 2016 Copyright © The American Society of Gene & Cell Therapy