- Email: [email protected]
711. Intravenous Neonatal Gene Therapy Corrects GM2 Gangliosidosis in Sandhoff Mice for Long-Term, By Using AAV Viral Vector Expressing a New Hexosaminidase Variant
Neurologic Diseases (Including Ophthalmic and Auditory Diseases) III a large portion of patients receiving L-DOPA treatment develop a series of debilitating hyperkinetic and dystonic movements known as L-DOPA-induced dyskinesias (LIDs). The molecular mechanisms behind LID development are unclear, as well as why some patients are resistant to LIDs and never acquire these symptoms. In an attempt to identify transcriptional differences between LID responders and LID non-responders in the rat parkinsonian 6-hydroxydopamine (6-OHDA) model, we performed a full genome array to identify differential transcript expression between these two groups. The orphan nuclear receptor Nurr1 was one transcript that was expressed at significantly higher levels—greater than 30 fold—in LID+ animals when compared to LID- animals. In the current study we sought to determine whether Nurr1 expression both promotes, and is required for, LID formation. Adult male Sprague-Dawley rats were rendered parkinsonian using6-OHDA. Animals that did not show a difference in paw use compared to a pre-lesion state were removed from the study one month later. At this time, remaining animals received a striatal injection of recombinant adeno-associated virus (rAAV) type 2/5 overexpressing either: 1) Nurr1 (or GFP as a control)(5x1013 vector genomes (vg)/ml) in order to test whether Nurr1 overexpression causes LIDs or 2) A shRNA targeting Nurr1 (or a titer-matched scrambled shRNA control)(2x1013 vg/ml) in order to test whether Nurr1 is required for LIDs. Four weeks following the vector injection, animals received escalating doses (0mg/kg - 24mg.kg) of L-DOPA every other day (M-Fr), and were evaluated for LIDs at 25 min intervals for 2-4 hours using the abnormal involuntary movement (AIM) rating scale. At doses equal to or exceeding 12mg/kg, Nurr1 overexpressing animals exhibited both more severe and longer lasting LIDs than their rAAV-GFP control counterparts. Conversely, dyskinesias were attenuated in animals receiving Nurr1-shRNA up to 18mg/kg of L-DOPA. In addition, postmortem analysis of striatal tissue revealed that the overexpression of the Nurr1 shRNA effectively inhibited the induction of L-DOPA induced Nurr1. These results demonstrate that overexpression of Nurr1 in the DA-depleted striatum of parkinsonian rats has a causative role in the expression of LIDs. Further, our data show that Nurr1 silencing in the striatum effectively inhibits the LID development. These data suggest that the maladaptive upregulation of Nurr1 in striatal neurons is a core event in the formation of LIDs. Consequently, targeting striatal Nurr1 activity may represent a novel therapeutic modality in the treatment of LIDs.
710. Optimization of AAV Vector Design for Safe Expression of β-N-Acetylhexosaminidase in the Brain for Tay-Sachs Disease Gene Therapy
Diane Golebiowski,1 Keiko Petrosky,2 Kajo Van der Marel,1 Elizabeth Hutto,2 Elizabeth Curran,2 Elena Balkanska-Sinclair,1 Nina Bishop,1 Rosemary Santos,1 Sheila Cummings Macri,2 Douglas Martin,3 Matthew Gounis,1 Julie Pilitsis,4 Wael Asaad,5 Miguel Sena-Esteves.1 1 University of Massachusetts Medical School, Worcester, MA; 2 New England Primate Research Center, Harvard Medical School, Southborough, MA; 3Scott-Ritchey Research Center, Auburn University, Auburn, AL; 4Neurosurgery, Albany Medical College, Albany, NY; 5Neurosurgery, Rhode Island Hospital and Brown University Alpert Medical School, Providence, RI.
The GM2 gangliosidoses are lysosomal storage disorders that encompass both Tay-Sachs and Sandhoff diseases. These diseases are associated with deficiencies in the lysosomal enzyme β-N-acetylhexosaminidase (HexA). These deficiencies result in accumulation of GM2 ganglioside (GM2) in the central nervous system leading to neuronal dysfunction and death. HexA is a heterodimer composed of α and β subunits encoded by HEXA and Molecular Therapy Volume 23, Supplement 1, May 2015 Copyright © The American Society of Gene & Cell Therapy
HEXB genes respectively. Gene therapy approaches using direct injection of AAV vectors into the brain of both small and large disease models (mice, cats, and sheep) have all been successful in treating CNS pathology as well as extending lifespan. These studies utilized two AAVrh8 vectors encoding species-specific alpha and beta subunits of HexA under a CBA promoter with a WPRE (CBAHexA-WPRE). However, when preclinical safety studies were performed in cynomolgus macaques (cm) using the same strategy, severe neurotoxicity was observed for doses 0.1-3.2E12 vg, which are comparable to those tested in other species on a vg/kg brain weight basis. We hypothesized that the cause of unexpected toxicity was due to high expression of HexA. In order to reduce expression of HexA while maintaining our AAV dose (1.78E12-5.34E13 vg/kg brain weight), we generated a series of new vectors with different combinations of promoters and expression elements with a gradient of HexA expression levels. We tested 7 designs of AAVrh8 vectors encoding cm HexA subunits in athymic nude mice (3.3 E13 vg/kg brain weight). In mice injected with the original vector, AAVrh8CBA-cmHexA-WPRE, we observed increased levels of reactive astrocytes (GFAP) and activated microglia (Iba1) at the injection site. We used this as a screen to test the other vectors for lower gliosis while expressing HexA activity above normal. Three vector designs emerged and were tested in cynomolgus macaques (n=2, 90 days) infused bilaterally into the thalamus and cerebral lateral ventricle at the intermediate dose (5.34 E12 vg/kg brain weight) as in the first study. The behavior of all monkeys remained normal throughout the study. An abnormal T2 weighted MRI signal was documented at day 90 post-injection in one injection site in a monkey in the cohort with the highest HexA activity (up to 88 fold over normal). This signal was absent in day 30 and 60 brain MRI. Neurohistopathological examination revealed considerable neurodegeneration at this site. The other two cohorts had minimal to no neurotoxicity associated with increased HexA expression (up to 9 fold). Two new AAV vectors have been identified for safe overexpression of HexA in the primate brain.
711. Intravenous Neonatal Gene Therapy Corrects GM2 Gangliosidosis in Sandhoff Mice for Long-Term, By Using AAV Viral Vector Expressing a New Hexosaminidase Variant
Karlaina J.L. Osmon,1 Evan Woodley,2 Patrick Thompson,3 Katalina Ong,3 Subha Karumuthil-Melethil,4 Brian Mark,5 Don Mahuran,6,7 Steven J. Gray,4,8 Jagdeep S. Walia.1,2,3 1 Centre for Neuroscience Research, Queen’s University, Kingston, ON, Canada; 2Department of Biomedical and Molecular Sciences, Queen’s University, Kingston, ON, Canada; 3Medical Genetics/ Departments of Pediatrics and Pathology and Molecular Medicine, Queen’s University, Kingston, ON, Canada; 4Gene Therapy Centre, University of North Carolina, Chapel Hill, NC; 5 Department of Microbiology, University of Manitoba, Winnepeg, MB, Canada; 6Genetics and Genome Biology, Sick Kids, Toronto, ON, Canada; 7Department of Laboratory Medicine and Pathology, University of Toronto, Toronto, ON, Canada; 8Department of Ophthalmology, University of North Carolina, Chapel Hill, NC. GM2 gangliosidosis is a group of neurodegenerative disorders, characterized by the malfunctioning Hexosaminidase A (HexA) enzyme, for which there is no treatment. HexA is composed of two similar, but non-identical subunits, the alpha and the beta, which must interact with the GM2 activator protein, a substrate-specific cofactor, to hydrolyze GM2. Mutation in either subunit (or the activator) results in the development of GM2 gangliosidosis. In these diseases, the malfunctioning protein is unable to play its role in cleaving GM2 ganglioside, whose accumulation within the neurons of the central nervous system is ultimately toxic. The resulting neuronal death induces the primary symptoms of the disease; motor impairment, S283
Neurologic Diseases (Including Ophthalmic and Auditory Diseases) III seizures, and sensory impairments. The aim of this study is to observe the long-term in vivo effects of a novel treatment in a Sandhoff (beta deficient) mouse model. The treatment utilized a new Hex isoenzyme, Hex M, which functions as a homodimer in the treatment of GM2 gangliosidosis. The HexM subunit is a variant of the human Hex alpha subunit containing critical beta-components that allow it to form stable homodimers and interact with the GM2 activator protein to reduce substrate storage. Our methods include intravenous injections of the neonatal mice with a self-complementary vector (with a synthetic promoter) expressing HexM at day 0-1. We monitored one cohort for 8 weeks and another cohort long-term (>40 weeks) for biochemical, behavioural and molecular analyses. Through the enzymatic and GM2 ganglioside lipid analyses, we see that with a slight increase in enzyme activity, there is a significant increase in the clearance of GM2 gangliosides. On behavioural tests, the treated mice outperform their knockout age matched controls. While the untreated controls die before the age of 15 weeks, treated animals have survived to more than 40 weeks and are still being monitored. The molecular analyses reveal a uniform distribution of the vector between brain and spinal cord regions. In conclusion, the neonatal delivery of our newly synthesized viral vector expressing HexM to the Sandhoff mice provided long-term correction of the disease. This study will have implications not only for treatment of Sandhoff, but also Tay-Sachs disease (alpha deficiency).
712. Improved Reduction in GM2 Ganglioside Accumulation in Tay-Sachs Mice Using a New Hexosaminidase Variant
Subha Karumuthil-Melethil,1 Patrick Thompson,2 Jagdeep S. Walia,2 Brian Mark,3 Don Mahuran,4,5 Steven J. Gray.1,6 1 Gene Therapy Center, U of North Carolina, Chapel Hill, NC; 2 Medical Genetics/Depts of Pediatrics and Pathology and Molecular Medicine, Queen’s Univ., Kingston, ON, Canada; 3Dept of Microbiolgy, U of Manitoba, Winnipeg, Canada; 4Genetics and Genome Biology, SickKids, Toronto, Canada; 5Dept of Lab. Medicine and Pathology, U of Toronto, Toronto, Canada; 6Dept of Ophthalmology, UNC, Chapel Hill, NC. GM2 gangliosidosis is a family of three genetic neurodegenerative disorders caused by the accumulation of GM2 gangliosides (GM2). Two of these are due to the deficiency of one of 2 similar but nonidentical subunits that comprise heterodimeric β-hexosaminidase A (HexA) which hydrolyzes GM2. Mutations in the α-subunit (encoded by HEXA) of the enzyme HexA lead to Tay-Sachs disease (TSD), wherein mutations in the β-subunit (encoded by HEXB) lead to Sandhoff disease (SD). In their acute infantile forms, both rapidly progress with fatal neurological deterioration during childhood. The most significant pathological feature of TSD and SD is GM2 accumulation in neurons. Since functional HexA is a heterodimer of the α- and β-subunits, the efficacy of overexpressing only the deficient subunit in a gene therapy approach is limited by the levels of the endogenous subunit. An effective approach to treat either TSD or SD would be to express the α- and β-subunits at equimolar ratios from the same vector, which for AAV has been limited by size restrictions. The present study used a new variant of the Hex α-subunit, containing critical sequences from the β-subunit that can form a stable homodimer (HexM) capable of hydrolyzing GM2. A selfcomplementary (sc) AAV genome was designed with a synthetic promoter to allow packaging of HEXM. To test the efficacy of HEXM compared to that of the unmodified HEXA, these were packaged into scAAV9 vectors and injected stereotaxically into 4 or 15 month old TSD mice along with an identical titer of scAAV9/GFP vector to track vector spread. The mice were euthanized after 4 weeks and brain sections were subjected to IHC analysis against GFP and GM2. The HexA-like activity was assessed by clearance of GM2 within S284
the injected region, compared to the contralateral brain hemisphere. Qualitatively, a marked reduction of GM2 was apparent in the areas of highest GFP expression. The various Hex vectors showed a clear difference in their ability to degrade GM2. As predicted, human HEXM was more capable of clearing GM2 aggregates than human HEXA at either 4 or 15 months of age. Interestingly, mouse HEXA was more effective than human HEXA, indicating a speciesincompatibility, presumably in the formation of a human α- and murine β-subunit heterodimer. This incompatibility further reinforces the utility of HexM to form a functional homodimer independent of the endogenous α- and β-subunit, which should be effective in treating both TSD and SD. In conclusion, modified HEXM can form a functional homodimer capable of clearing GM2 aggregates. It can be packaged in a scAAV9 vector, which is amenable for strategies directed at widespread CNS gene transfer. These technological advances overcome previous barriers and provide pivotal reagents to develop a translatable gene therapy for TSD and SD.
713. IntraCSF Administration of AAV9/ MeCP2 Extends Lifespan of MeCP2-Null Mice While Preventing Toxicity Associated With IV Administration
Sarah E. Sinnett,1 Sahana N. Kalburgi,2 Steven J. Gray.1 1 Gene Therapy Center, University of North Carolina at Chapel Hill, Chapel Hill, NC; 2Biomedical Research Education and Training, Vanderbilt University, Nashville, TN. Rett Syndrome (RTT) is a neurodevelopmental disorder that shortens the lifespan of patients while depriving them of the ability to walk, talk, or interact with others. RTT is caused by inactivating mutations in the X chromosome-linked gene encoding methylCpG-binding protein 2 (MeCP2), a transcription regulator that is highly expressed in neurons. Patients with RTT express mutant MeCP2 in ∼50% of their cells due to random X-chromosome inactivation in each cell. The remaining “healthy” cells have an activated chromosome encoding functional (WT) MeCP2. Although researchers have already used MeCP2 gene therapy to reverse the RTT phenotype in mosaic mice, concerns about side effects (i.e., motor and learning deficits) resulting from MeCP2 overexpression in “healthy” cells persist. Indeed, humans suffering from MeCP2 duplication syndrome have been shown to suffer from muscular spasticity and intellectual disabilities. In addition to persistent concerns about MeCP2 overexpression, IV administration of the scAAV9/hMeCP2 vector (1x1011 vg/mouse) has been shown to cause significant liver toxicity in MeCP2-null mice (Gadalla et al, Mol Ther, 2013). We tested the hypothesis that by moving from an IV route of administration to an intraCSF route, we could lower the dose of AAV vector enough to avoid the high peripheral organ transduction that generates toxic side effects. Modeling our earlier experiment with an IV route, juvenile (4-5 week old) knock-out (KO) mice and WT littermates received a single intrathecal injection of AAV9/ MeCP2 vector. RTT mice dosed intrathecally with 1x1010 vg had a 48% increase in median survival compared to vehicle-injected mice (n=6 treated; n=11 vehicle; P=0.008), nearly identical to the rescue we previously observed following the IV approach but at 1/10th the dose and avoiding overexpression-related toxicity in the liver. In conclusion, by changing the route of administration of the AAV9/ MeCP2 vector from IV to intraCSF, we were able to generate a nearly identical therapeutic outcome with a 10-fold lower dose, and without excessive (toxic) gene transfer to peripheral organs. As a next step to utilize the intraCSF route of administration, DNA shuffling and directed evolution were utilized to select for AAV capsid variants that would have higher efficiency and improved biodistribution in the RTT mice after intraCSF administration. Multiple clones were selected Molecular Therapy Volume 23, Supplement 1, May 2015 Copyright © The American Society of Gene & Cell Therapy
710. Optimization of AAV Vector Design for Safe Expression of β-N-Acetylhexosaminidase in the Brain for Tay-Sachs Disease Gene Therapy
Diane Golebiowski,1 Keiko Petrosky,2 Kajo Van der Marel,1 Elizabeth Hutto,2 Elizabeth Curran,2 Elena Balkanska-Sinclair,1 Nina Bishop,1 Rosemary Santos,1 Sheila Cummings Macri,2 Douglas Martin,3 Matthew Gounis,1 Julie Pilitsis,4 Wael Asaad,5 Miguel Sena-Esteves.1 1 University of Massachusetts Medical School, Worcester, MA; 2 New England Primate Research Center, Harvard Medical School, Southborough, MA; 3Scott-Ritchey Research Center, Auburn University, Auburn, AL; 4Neurosurgery, Albany Medical College, Albany, NY; 5Neurosurgery, Rhode Island Hospital and Brown University Alpert Medical School, Providence, RI.
The GM2 gangliosidoses are lysosomal storage disorders that encompass both Tay-Sachs and Sandhoff diseases. These diseases are associated with deficiencies in the lysosomal enzyme β-N-acetylhexosaminidase (HexA). These deficiencies result in accumulation of GM2 ganglioside (GM2) in the central nervous system leading to neuronal dysfunction and death. HexA is a heterodimer composed of α and β subunits encoded by HEXA and Molecular Therapy Volume 23, Supplement 1, May 2015 Copyright © The American Society of Gene & Cell Therapy
HEXB genes respectively. Gene therapy approaches using direct injection of AAV vectors into the brain of both small and large disease models (mice, cats, and sheep) have all been successful in treating CNS pathology as well as extending lifespan. These studies utilized two AAVrh8 vectors encoding species-specific alpha and beta subunits of HexA under a CBA promoter with a WPRE (CBAHexA-WPRE). However, when preclinical safety studies were performed in cynomolgus macaques (cm) using the same strategy, severe neurotoxicity was observed for doses 0.1-3.2E12 vg, which are comparable to those tested in other species on a vg/kg brain weight basis. We hypothesized that the cause of unexpected toxicity was due to high expression of HexA. In order to reduce expression of HexA while maintaining our AAV dose (1.78E12-5.34E13 vg/kg brain weight), we generated a series of new vectors with different combinations of promoters and expression elements with a gradient of HexA expression levels. We tested 7 designs of AAVrh8 vectors encoding cm HexA subunits in athymic nude mice (3.3 E13 vg/kg brain weight). In mice injected with the original vector, AAVrh8CBA-cmHexA-WPRE, we observed increased levels of reactive astrocytes (GFAP) and activated microglia (Iba1) at the injection site. We used this as a screen to test the other vectors for lower gliosis while expressing HexA activity above normal. Three vector designs emerged and were tested in cynomolgus macaques (n=2, 90 days) infused bilaterally into the thalamus and cerebral lateral ventricle at the intermediate dose (5.34 E12 vg/kg brain weight) as in the first study. The behavior of all monkeys remained normal throughout the study. An abnormal T2 weighted MRI signal was documented at day 90 post-injection in one injection site in a monkey in the cohort with the highest HexA activity (up to 88 fold over normal). This signal was absent in day 30 and 60 brain MRI. Neurohistopathological examination revealed considerable neurodegeneration at this site. The other two cohorts had minimal to no neurotoxicity associated with increased HexA expression (up to 9 fold). Two new AAV vectors have been identified for safe overexpression of HexA in the primate brain.
711. Intravenous Neonatal Gene Therapy Corrects GM2 Gangliosidosis in Sandhoff Mice for Long-Term, By Using AAV Viral Vector Expressing a New Hexosaminidase Variant
Karlaina J.L. Osmon,1 Evan Woodley,2 Patrick Thompson,3 Katalina Ong,3 Subha Karumuthil-Melethil,4 Brian Mark,5 Don Mahuran,6,7 Steven J. Gray,4,8 Jagdeep S. Walia.1,2,3 1 Centre for Neuroscience Research, Queen’s University, Kingston, ON, Canada; 2Department of Biomedical and Molecular Sciences, Queen’s University, Kingston, ON, Canada; 3Medical Genetics/ Departments of Pediatrics and Pathology and Molecular Medicine, Queen’s University, Kingston, ON, Canada; 4Gene Therapy Centre, University of North Carolina, Chapel Hill, NC; 5 Department of Microbiology, University of Manitoba, Winnepeg, MB, Canada; 6Genetics and Genome Biology, Sick Kids, Toronto, ON, Canada; 7Department of Laboratory Medicine and Pathology, University of Toronto, Toronto, ON, Canada; 8Department of Ophthalmology, University of North Carolina, Chapel Hill, NC. GM2 gangliosidosis is a group of neurodegenerative disorders, characterized by the malfunctioning Hexosaminidase A (HexA) enzyme, for which there is no treatment. HexA is composed of two similar, but non-identical subunits, the alpha and the beta, which must interact with the GM2 activator protein, a substrate-specific cofactor, to hydrolyze GM2. Mutation in either subunit (or the activator) results in the development of GM2 gangliosidosis. In these diseases, the malfunctioning protein is unable to play its role in cleaving GM2 ganglioside, whose accumulation within the neurons of the central nervous system is ultimately toxic. The resulting neuronal death induces the primary symptoms of the disease; motor impairment, S283
Neurologic Diseases (Including Ophthalmic and Auditory Diseases) III seizures, and sensory impairments. The aim of this study is to observe the long-term in vivo effects of a novel treatment in a Sandhoff (beta deficient) mouse model. The treatment utilized a new Hex isoenzyme, Hex M, which functions as a homodimer in the treatment of GM2 gangliosidosis. The HexM subunit is a variant of the human Hex alpha subunit containing critical beta-components that allow it to form stable homodimers and interact with the GM2 activator protein to reduce substrate storage. Our methods include intravenous injections of the neonatal mice with a self-complementary vector (with a synthetic promoter) expressing HexM at day 0-1. We monitored one cohort for 8 weeks and another cohort long-term (>40 weeks) for biochemical, behavioural and molecular analyses. Through the enzymatic and GM2 ganglioside lipid analyses, we see that with a slight increase in enzyme activity, there is a significant increase in the clearance of GM2 gangliosides. On behavioural tests, the treated mice outperform their knockout age matched controls. While the untreated controls die before the age of 15 weeks, treated animals have survived to more than 40 weeks and are still being monitored. The molecular analyses reveal a uniform distribution of the vector between brain and spinal cord regions. In conclusion, the neonatal delivery of our newly synthesized viral vector expressing HexM to the Sandhoff mice provided long-term correction of the disease. This study will have implications not only for treatment of Sandhoff, but also Tay-Sachs disease (alpha deficiency).
712. Improved Reduction in GM2 Ganglioside Accumulation in Tay-Sachs Mice Using a New Hexosaminidase Variant
Subha Karumuthil-Melethil,1 Patrick Thompson,2 Jagdeep S. Walia,2 Brian Mark,3 Don Mahuran,4,5 Steven J. Gray.1,6 1 Gene Therapy Center, U of North Carolina, Chapel Hill, NC; 2 Medical Genetics/Depts of Pediatrics and Pathology and Molecular Medicine, Queen’s Univ., Kingston, ON, Canada; 3Dept of Microbiolgy, U of Manitoba, Winnipeg, Canada; 4Genetics and Genome Biology, SickKids, Toronto, Canada; 5Dept of Lab. Medicine and Pathology, U of Toronto, Toronto, Canada; 6Dept of Ophthalmology, UNC, Chapel Hill, NC. GM2 gangliosidosis is a family of three genetic neurodegenerative disorders caused by the accumulation of GM2 gangliosides (GM2). Two of these are due to the deficiency of one of 2 similar but nonidentical subunits that comprise heterodimeric β-hexosaminidase A (HexA) which hydrolyzes GM2. Mutations in the α-subunit (encoded by HEXA) of the enzyme HexA lead to Tay-Sachs disease (TSD), wherein mutations in the β-subunit (encoded by HEXB) lead to Sandhoff disease (SD). In their acute infantile forms, both rapidly progress with fatal neurological deterioration during childhood. The most significant pathological feature of TSD and SD is GM2 accumulation in neurons. Since functional HexA is a heterodimer of the α- and β-subunits, the efficacy of overexpressing only the deficient subunit in a gene therapy approach is limited by the levels of the endogenous subunit. An effective approach to treat either TSD or SD would be to express the α- and β-subunits at equimolar ratios from the same vector, which for AAV has been limited by size restrictions. The present study used a new variant of the Hex α-subunit, containing critical sequences from the β-subunit that can form a stable homodimer (HexM) capable of hydrolyzing GM2. A selfcomplementary (sc) AAV genome was designed with a synthetic promoter to allow packaging of HEXM. To test the efficacy of HEXM compared to that of the unmodified HEXA, these were packaged into scAAV9 vectors and injected stereotaxically into 4 or 15 month old TSD mice along with an identical titer of scAAV9/GFP vector to track vector spread. The mice were euthanized after 4 weeks and brain sections were subjected to IHC analysis against GFP and GM2. The HexA-like activity was assessed by clearance of GM2 within S284
the injected region, compared to the contralateral brain hemisphere. Qualitatively, a marked reduction of GM2 was apparent in the areas of highest GFP expression. The various Hex vectors showed a clear difference in their ability to degrade GM2. As predicted, human HEXM was more capable of clearing GM2 aggregates than human HEXA at either 4 or 15 months of age. Interestingly, mouse HEXA was more effective than human HEXA, indicating a speciesincompatibility, presumably in the formation of a human α- and murine β-subunit heterodimer. This incompatibility further reinforces the utility of HexM to form a functional homodimer independent of the endogenous α- and β-subunit, which should be effective in treating both TSD and SD. In conclusion, modified HEXM can form a functional homodimer capable of clearing GM2 aggregates. It can be packaged in a scAAV9 vector, which is amenable for strategies directed at widespread CNS gene transfer. These technological advances overcome previous barriers and provide pivotal reagents to develop a translatable gene therapy for TSD and SD.
713. IntraCSF Administration of AAV9/ MeCP2 Extends Lifespan of MeCP2-Null Mice While Preventing Toxicity Associated With IV Administration
Sarah E. Sinnett,1 Sahana N. Kalburgi,2 Steven J. Gray.1 1 Gene Therapy Center, University of North Carolina at Chapel Hill, Chapel Hill, NC; 2Biomedical Research Education and Training, Vanderbilt University, Nashville, TN. Rett Syndrome (RTT) is a neurodevelopmental disorder that shortens the lifespan of patients while depriving them of the ability to walk, talk, or interact with others. RTT is caused by inactivating mutations in the X chromosome-linked gene encoding methylCpG-binding protein 2 (MeCP2), a transcription regulator that is highly expressed in neurons. Patients with RTT express mutant MeCP2 in ∼50% of their cells due to random X-chromosome inactivation in each cell. The remaining “healthy” cells have an activated chromosome encoding functional (WT) MeCP2. Although researchers have already used MeCP2 gene therapy to reverse the RTT phenotype in mosaic mice, concerns about side effects (i.e., motor and learning deficits) resulting from MeCP2 overexpression in “healthy” cells persist. Indeed, humans suffering from MeCP2 duplication syndrome have been shown to suffer from muscular spasticity and intellectual disabilities. In addition to persistent concerns about MeCP2 overexpression, IV administration of the scAAV9/hMeCP2 vector (1x1011 vg/mouse) has been shown to cause significant liver toxicity in MeCP2-null mice (Gadalla et al, Mol Ther, 2013). We tested the hypothesis that by moving from an IV route of administration to an intraCSF route, we could lower the dose of AAV vector enough to avoid the high peripheral organ transduction that generates toxic side effects. Modeling our earlier experiment with an IV route, juvenile (4-5 week old) knock-out (KO) mice and WT littermates received a single intrathecal injection of AAV9/ MeCP2 vector. RTT mice dosed intrathecally with 1x1010 vg had a 48% increase in median survival compared to vehicle-injected mice (n=6 treated; n=11 vehicle; P=0.008), nearly identical to the rescue we previously observed following the IV approach but at 1/10th the dose and avoiding overexpression-related toxicity in the liver. In conclusion, by changing the route of administration of the AAV9/ MeCP2 vector from IV to intraCSF, we were able to generate a nearly identical therapeutic outcome with a 10-fold lower dose, and without excessive (toxic) gene transfer to peripheral organs. As a next step to utilize the intraCSF route of administration, DNA shuffling and directed evolution were utilized to select for AAV capsid variants that would have higher efficiency and improved biodistribution in the RTT mice after intraCSF administration. Multiple clones were selected Molecular Therapy Volume 23, Supplement 1, May 2015 Copyright © The American Society of Gene & Cell Therapy