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22 Combination Therapy for the Treatment of Infantile Neuronal Ceroid Lipofuscinosis (INCL) Molecular Therapy Volume 18, Supplement 1, May 2010 Copyright © The American Society of Gene & Cell Therapy[.]
NEUROLOGIC & OPHTHALMIC GENE & CELL THERAPY 20 Neonatal Intracerebroventricular Infusion of AAV2/8 Vector Leads to High Levels of Human Į-L-Iduronidase Expression in the Brain and Complete Neurophenotypic Correction in a Murine Model of Mucopolysaccharidosis Type I Daniel A Wolf,1 Andrew W Lenander,1 Zhenhong Nan,2 Lalitha R Belur,1 Debra Swanson,1 Chester B Whitley,1 Walter C Low,2 R Scott McIvor.1 Genetics, Cell Biology, and Development, University of Minnesota, Minneapolis, MN; 2Neurosurgery, University of Minnesota, Minneapolis, MN Mucopolysaccharidosis type I (MPS I) is an autosomal recessive inherited disorder caused by deciency of the glycosidase α-Liduronidase (IDUA) IDUA is required for the degradation of the glycosaminoglycans (GAG) heparan sulfate and dermatan sulfate, and deciency of the enzyme leads to lysosomal storage of these substrates Manifestations of severe MPS I begin to develop within the rst two years of life and include growth delay, hepatosplenomegaly, skeletal deformities, increased urinary GAG, corneal clouding, and neurological defects Current therapies for MPS I include allogeneic hematopoietic stem cell transplantation and enzyme replacement therapy However, the neurological effectiveness of these therapies is limited by inability of the enzyme to efciently transverse the blood-brain-barrier To address this issue, we have tested direct infusion of AAV vectors into the brain as an alternative approach to treatment Here we report our results involving intracerebroventricular infusion of an AAV2/8 vector encoding human IDUA under the control of a shortened CAGS promoter x 1010 vector genomes were stereotactically injected into the right-side lateral ventricle of newborn, 6-week-old, and 7-month-old Idua-/- mice Neonatally treated animals demonstrated complete correction in a Morris water maze test of spatial navigation and memory at months of age Animals treated at weeks of age displayed partial correction of the learning decit, while animals treated at months of age displayed no improvement The animals were perfused and sacriced at 10 months of age and their brains were microdissected for further analysis Quantitative PCR using primers specic to the hIDUA gene demonstrated widespread vector distribution throughout the brain of neonatally treated mice, with the highest levels achieved in the hippocampus These animals displayed extremely high levels of IDUA throughout the brain; in particular, the hippocampus of treated animals contained IDUA levels 24-fold higher than wild-type controls The distribution of AAV transduction and IDUA activity was less pronounced in animals treated at weeks and months of age Immunouorescence analysis using an anti-hIDUA antibody revealed intense staining in multiple structures throughout the brain of animals treated at birth with a large concentration of labeling present in structures of the limbic system Less intense staining was observed in brain sections from animals treated at weeks and months of age with much of the cell labeling occurring in structures adjacent to the lateral ventricles These results show that the CNS manifestations of MPS I disease can be completely prevented by ICV infusion of AAV2/8 vector in newborn animals Furthermore, these data suggest that age at the time of treatment and the level and distribution of enzyme expressed are important determinants for the effectiveness of treatment 21 Circuit-Specic Expression of Channelrhodopsin Restores Visual Function in Blind rd1, rd16, and rho -/- Mice Alan Horsager,1,2 Jian Wen Liu,3 Edward S Boyden,1,4 A Cyrus Arman,2 Kimberly A Silka,2 Mehdi Doroudchi,1 Shannon E Boye,3 Benjamin C Matteo,1 Alapakkam P Sampath,2 William W Hauswirth.3 Eos Neuroscience, Inc., Los Angeles, CA; 2Zilkha Neurogenetic Institute, University of Southern California, Los Angeles, CA; Ophthalmology, University of Florida, Gainesville, FL; 4Media Lab, Massachusetts Institute of Technology, Cambridge, MA Purpose: Channelrhodopsin-2 (ChR2) is a light-sensitive protein that, when expressed in neurons, depolarizes the cell in response to light stimulation Microelectronic neural prostheses result in broad and indiscriminant stimulation of the neural interface, whereas expression of ChR2 can be genetically-targeted using cell typespecic regulatory sequences (i.e., promoters) such that activation of specic neural circuits can be achieved Using the GRM6 regulatory sequence in combination with a tyrosine-mutated adeno-associated virus (AAV), we were able to target expression of ChR2 to the ON bipolar cells of the retina using either a subretinal or intravitreal injection, and subsequently restore visual function in multiple mouse models of blindness Methods: We evaluated retinal bipolar cell transduction using wild-type and capsid tyrosine-mutated AAV serotypes Vector, including the ChR2 and green uorescent protein (GFP) genes, was either subretinally or intravitreally injected in rd1, rd16, and rho -/- mice under the control of the GRM6 promoter Expression and localization of the ChR2-GFP fused protein was evaluated using confocal microscopy and immunohistochemistry Visual function was measured behaviorally in wild-type, untreated, and ChR2-treated mice using a water maze and retinal patch clamp recordings, respectively Results: Both wild-type and mutated serotypes were effective at transducing retinal bipolar cells The capsid tyrosine-mutated serotypes were able to increase bipolar cell transduction by as much as 20-fold, even with an intravitreal injection In the water maze task, the ChR2-treated mice learned the task nearly as well as the wild-type mice (the untreated mice were unable to learn the task) Additionally, the light intensity necessary to restore this visually-guided behavior was within the normal visual dynamic range of human vision Conclusions: Targeted expression of ChR2 in retinal ON bipolar cells restores circuit-specic computation, behavioral, and physiological visual function in all treated mice, suggesting the broad applicability of this gene therapy Equally as important, we can target ChR2 expression to bipolar cells using a capsid tyrosinemutated AAV, even with an intravitreal injection Further research is necessary to evaluate visual acuity and methods of increasing the sensitivity in these treated animals, taking into account new variants of ChR2 Recent bioengineering work from our group in nonhuman primates suggests hope for a translational path 22 Combination Therapy for the Treatment of Infantile Neuronal Ceroid Lipofuscinosis (INCL) Shannon L Macauley-Rambach,1 Marie S Roberts,1 Mark S Sands.1 Internal Medicine, Washington University School of Medicine, Saint Louis, MO Infantile neuronal ceroid lipofuscinosis (INCL) is a rapidly progressing, inherited neurodegenerative lysosomal storage disease (LSD) INCL is caused by mutations in the CLN1 gene leading to a deciency in the lysosomal enzyme, palmitoyl protein thioesterase (PPT1) Pathological changes in the central nervous system (CNS) include brain atrophy, cortical thinning, autouorescent accumulation, neurodegeneration, and glial activation The CNS pathology correlates with clinical signs such as seizures, blindness, motor dysfunction, Molecular Therapy Volume 18, Supplement 1, May 2010 Copyright © The American Society of Gene & Cell Therapy S9 NEUROLOGIC & OPHTHALMIC GENE & CELL THERAPY and mental retardation Invariably, patients die by yr of age A murine model of INCL, the PPT1-/- mouse, was created that mimics the human disease PPT1-/- mice have a decreased life span, visual decits, cognitive decline, epilepsy, and motor abnormalities Histologically, PPT1-/- brains show similar pathological changes as children with INCL including autouorescent accumulation, neurodegeneration, glial activation, and brain atrophy Therefore, the PPT1-/- mouse is an excellent model to investigate therapeutic options for the treatment of INCL To date, both gene- and stem cell- based therapies have been explored as possible therapies for INCL Although both therapeutic options demonstrated some benets in terms of histological parameters, the behavioral improvements were modest and no improvement in lifespan was achieved Thus, we hypothesized that several therapeutic modalities must be used in concert to achieve the maximum efcacy in INCL Utilizing various conformers of puried SYN and a DA quinone modied peptide to screen a combinatorial phage display library expressing human immunoglobulin heavy and light chain variable regions we have identied conformation-specic SYN-specic scFvs (SYNscFvs) These scFvs have been engineered for expression using a rAAV platform Here we demonstrate that stereotaxic injection of rAAVSYNscFvs and rAAVDAQscFv into the SN of transgenic mice that overexpress human SYN attenuates SNpc DAN loss rAAVscFvs directed against SYN-specic conformers will allow us to further investigate the role of SYN aggregation in the pathogenesis of PD and represent a potentially novel therapeutic approach for this disorder scFv directed against DA quinone modied proteins open up investigations of novel substrates and their roles in SYN mediated pathogenesis Supported by DOD #DAMD17-02-1-0695 to Howard J Federoff In this study, we used a combination approach for the treatment of INCL We previously demonstrated that intracranial delivery of six injections of adeno-associated virus expressing PPT1 (i.e AAV2PPT1) at birth in the PPT1-/- mice provided the greatest therapeutic promise Thus, we employed a similar approach in our current study but used AAV2/5 due to its greater transduction efciency and higher expression in the CNS Furthermore, we teamed this gene therapy approach with daily injections of phosphocystaemine (Cystagon) Cystagon is a lysosomotropic drug that disrupts thioester linkages in a manner similar to PPT1 Since Cystagon is safe, bioavailable, and able to cross the blood brain barrier, we hypothesized that daily injections of this drug would synergize with the intracranial AAV2/5 to provide greater therapeutic benet in PPT1-/- mice In fact, our preliminary ndings demonstrate that the dual-therapy is more benecial than either AAV2/5 or Cystagon alone While PPT1-/- mice have a 50% mortality by 33 wk of age, all of the dual-treated mice are still alive at 41 wk of age At 28 wk of age, dual-treated PPT1-/- mice have an increased brain mass compared to PPT1-/- mice Most importantly, the dual-treated mice were indistinguishable from normal mice on the constant speed and rocking rotorod tests out to 28 wk of age By 36 wk of age, motor function of the dual-treated group began to diminish but they are still performing better than any singularly treated group Our data suggest that intracranial AAV2/5 in combination with daily injections of Cystagon demonstrates great therapeutic promise for the treatment of INCL 24 Patterns of Motor Function Recovery in a Murine Model of Severe Menkes Disease Rescued by Brain-Directed AAV5 Gene Therapy Plus Copper 23 rAAV Delivery of Į-Synuclein-Specic Single Chain Antibodies Attenuates Dopamine Loss in a Mouse Model of Parkinson’s Disease Kathleen Maguire-Zeuss,1 William J Bowers,2 Wade Narrow,2 Eric Yehling,2 Howard J Federoff.1 Department of Neuroscience, Georgetown University Medical Center, Washington, DC; 2Department of Neurology, Center for Neural Development and Disease, University of Rochester School of Medicine and Dentistry, Rochester, NY Parkinson’s disease (PD) is a progressive neurodegenerative disorder triggered by disparate mechanisms The disease invariantly produces loss of dopamine neurons (DANs) in the substantia nigra pars compacta (SNpc) The hallmark pathobiologic feature of PD is the formation of intracytoplasmic α-synuclein-containing inclusions called as Lewy bodies which accumulate in the remaining SNpc DANs The selective insult to the dopamine (DA) neuron is posited to involve oxidative injury mediated in part through the formation of DA adducts made onto α-synuclein (SYN) and perhaps other proteins We hypothesize that therapies specically designed to interact and reduce toxic SYN conformers will attenuate disease progression We undertook a gene therapy approach whereby conformationspecic humanized single-chain antibodies (scFvs) are expressed and intended to interfere with misfolded and DA adducted proteins S10 Anthony Donsante,1 Stephen G Kaler.1 Unit on Human Copper Metabolism, Molecular Medicine Program, NICHD, NIH, Bethesda, MD Menkes disease (MD) is an inherited neurodegenerative disorder caused by mutations in the X-linked copper transporter, ATP7A Affected infants show reduced copper transport across the gut and the blood-brain barrier, resulting in decient activities of copperdependent enzymes, including some critical for proper neurological function MD patients exhibit neurodevelopmental delay, hypotonia, seizures and premature death When copper injection therapy is begun very early in life, improved outcomes are possible for patients whose mutant alleles retain residual copper transport activity (N Engl J Med 2008 358:605-614) However, in patients with severe mutations, even very early copper injection treatment fails to normalize neurological performance We previously reported the rescue of the mottled-brindled (mo-br) mouse, a model of severe MD, using intracerebroventricular AAV5-mediated gene therapy plus copper in 2-3 day old mice This combination of therapies extended median survival from 13 d (range 12-14) in untreated mice to 43 d, with 33% living beyond 110 d In addition, these mice showed substantial biochemical and neuropathological improvements Here we report the results of serial neurobehavioral phenotyping in long-surviving rescued mo-br mice, using the constant speed rotarod (a measure of balance and coordination), wire hang (to evaluate neuromuscular strength), and Morris Water Maze tests (to assess spatial learning) The rotarod and wire hang tests were performed every week beginning at 25 d of age (the oldest mice are now over 221 d of age; three trials per time point, 60 s maximum) Combination-treated mo-br mice were similar to wild type controls on the rotarod at 25 d of age [mo-br (N=10) 56 s, wild type (N=9) 60 s] However, they exhibited signicant impairment in subsequent weeks, averaging 15 to 20 s on the rod (p