589 CNS Administration of an AAVrh 10 Gene Transfer Vector Expressing the Human ARSA cDNA to Nonhuman Primates Provides Widespread Activity across the CNS Molecular Therapy Volume 21, Supplement 1, Ma[.]
NEUROLOGIC GENE & CELL THERAPY II expensive and inconvenient treatment approach To begin to explore a new potential therapeutic avenue for this disease, we constructed a NAGLU-IGF2 cDNA, and documented robust expression and NAGLU enzyme activity in transfected CHO and Hek293T cells We prepared recombinant adeno-associated virus serotype (rAAV5) containing the NAGLU-IGF2 cDNA with the cytomegalovirus enhancer/chicken -actin promoter to drive transgene expression We are performing a pilot in vivo study in normal adult rats using intracerebroventricular injections of 6×1010 rAAV5 viral particles, under stereotaxic guidance The rAAV5 serotype is known to target choroid plexus epithelia, specialized structures that project into the brain’s ventricular cavities and show limited cellular turnover Choroid plexuses produce CSF by transporting water and ions, and regulate the biochemical and protein composition of CSF AAV vector delivery to choroid plexus epithelia would provide a steady source of human NAGLU-IGF2 in the CSF to treat the brain Testing in MPS IIIB mice will evaluate the safety and efficacy of this approach to correct or prevent brain pathology by re-modeling choroid plexus to continuously secrete NAGLU-IGF2 This unique therapeutic approach seeks to combine the benefits of M6P-independent endocytosis and viral gene therapy, and may enable efficient NAGLUIGF2 distribution throughout central nervous system 587 Mitigation of DNA Damage Responses and Oxidative Stress in Cells with Impaired Telomerase Function Larisa Pereboeva,1 Erik Westin,2,3 Mary Gannon,5 Lawrence Lamb,1 Aloysius Klingelhutz,4 Frederick Goldman.2 Department of Medicine, Division of Hematology Oncology, University of Alabama at Birmingham, Birmingham, AL; Department of Pediatrics, Division of Hematology Oncology, Children’s Hospital of Alabama, University of Alabama at Birmingham, Birmingham, AL; 3Department of Biochemistry and Molecular Genetics, University of Alabama at Birmingham, Birmingham, AL; 4Department of Microbiology, University of Iowa, Iowa City, IA; 5Department of Genetics, University of Alabama at Birmingham, Birmingham, AL The ‘telomeropathies’ are a newly described group of human diseases with genetic mutations that decrease telomere length or telomerase activity One such disease, Dyskeratosis congenita (DC) is an inherited multisystem disorder characterized by premature aging, cancer predisposition, bone marrow failure, and critically shortened telomeres It is widely accepted that molecular pathology of DC cells stems from abnormal signaling from shortened telomeres However, exact mechanisms of this signaling are unknown and treatment strategies are limited The requirement of telomerase activity is most evident in highly proliferative cells, and those lacking this enzymatic activity, either as a consequence of the natural aging process of genetic mutation, demonstrate a growth disadvantage and features of senescence We have recently shown that DC cells also have a growth disadvantage, or stressed phenotype, that is associated with increased basal levels of intracellular oxidative stress, apoptosis and p53 expression We hypothesized that there is a causal relationship between diminished telomerase function and imbalances in steadystate levels of oxidative stress Therefore, restoration of telomerase activity by transduction with exogenous telomerase may correct the stressed phenotype of DC cells and normalize levels of ROS and p53 We have studied primary fibroblasts and lymphocytes derived from DC patients as cell types representative of DC pathology Growth disadvantage of both cell types in continuous cell culture compared to corresponding normal controls was documented Studies on DC fibroblasts revealed that heightened reactive oxygen species (ROS) and DNA damage response (DDR) levels can be reduced in two experimental conditions with a concomitant effect on cell growth First, culturing DC fibroblasts in low oxygen conditions (5% O2) Molecular Therapy Volume 21, Supplement 1, May 2013 Copyright © The American Society of Gene & Cell Therapy partially restored their proliferative capacity In addition, we were able to ameliorate the proliferative disadvantage in DC fibroblasts by introduction of exogenous TERT by way of transduction with a retroviral vector Importantly, expression of TERT reduced steadystate levels of ROS in DC cells Recently we have also uncovered elevated basal levels of ROS and p53 expression in DC lymphocytes These levels, as well as replicative lifespan, can be partially normalized by treatment with antioxidant N-acetyl cysteine (NAC) These data suggest a regulatory role of telomeres or telomerase activity in maintaining homeostatic intracellular levels of ROS and p53 588 Abstract Withdrawn Neurologic Gene & Cell Therapy II 589 CNS Administration of an AAVrh.10 Gene Transfer Vector Expressing the Human ARSA cDNA to Nonhuman Primates Provides Widespread Activity across the CNS Jonathan B Rosenberg,1 Dolan Sondhi,1 David G Rubin,1 Sébastien Monette,2 Alvin Chen,1 Sara A Cram,1 Benjamin Van de Graaf,1 Bishnu P De,1 Stephen M Kaminsky,1 Caroline Sevin,3,4 Patrick Aubourg,3,4 Ronald G Crystal.1 Weill Cornell Medical College, NY, NY; 2Tri-Institutional Laboratory of Comparative Pathology, Weill Cornell Medical College and Memorial Sloan-Kettering Cancer Center, NY, NY; INSERM U986, Université Paris-Descartes, Paris, France; Bicêtre Hospital, Paris, France Metachromatic leukodystrophy (MLD), a fatal lysosomal lipid storage disorder caused by the deficiency of the lysosomal enzyme, arylsulfatase A (ARSA), leads to an accumulation of sulfatides, causing a widespread demyelination in both the central (CNS) and peripheral (PNS) nervous systems MLD is a good candidate for gene therapy because the ARSA protein is secreted and is capable of cross correcting neighboring cells, i.e., it is not necessary to achieve gene transfer to every cell in the CNS Leveraging the knowledge that adeno-associated viruses (AAV) have demonstrated persistent and efficacious gene transfer in animal studies with excellent safety profiles in human clinical trials, and that the rhesus serotype AAVrh.10 expressing FLAG-tagged ARSA (AAVrh.10hARSAFLAG) expresses ARSA throughout the CNS of rodents, we compared several routes of delivery with this vector in nonhuman primates to determine which would provide the broadest distribution of ARSA enzyme Five routes of vector delivery were tested using a total dose of 1.5x1012 genome copies of AAVrh.10hARSA-FLAG for each route: (1) cortical delivery to the white matter (ventral to the cortex); (2) deep grey matter (putamen, thalamus, and caudate) plus the overlaying white matter; (3) convection enhanced delivery to same deep grey matter locations; (4) the lateral cerebral ventricle; and (5) intra-arterial delivery with hyperosmotic mannitol in the middle cerebral artery After 13 wk, the brains were analyzed for the distribution of ARSA protein and enzymatic activity The right hemisphere was subdivided into cm3 cubes, homogenized and assayed for functional ARSA activity The left hemisphere was fixed in formalin and sectioned for immunohistochemistry using the FLAG epitope The distribution of ARSA activity in each of the CNS (intracerebral) administered routes was significantly higher than PBS-administered controls, while the intraventricular and intra-arterial routes failed to demonstrate measurable levels above controls Immunohistochemistry using anti-FLAG antibodies confirmed these results with extensive ARSA expression from all CNS (intracerebral) administered routes of delivery with staining in the cortex, white matter, deep grey matter of the striatum, thalamus, choroid plexus and spinal cord dorsal root S225 NEUROLOGIC GENE & CELL THERAPY II ganglions Of these routes, the cortical administration generated the broadest distribution of ARSA with 80% of the brain cubes displaying more than a therapeutic (10%) increase in ARSA activity above PBS controls We conclude that AAVrh.10-mediated delivery of the ARSA transgene via cortical CNS administration into the white matter will lead to the widest distribution of ARSA in the CNS with likelihood of providing a therapeutic benefit for MLD patients 590 Intracranial AAV Gene Therapy Extends the Life Span of GM2 Gangliosidosis Cats >Four-Fold with No Clinical Evidence of Vector Toxicity Allison M Bradbury,1 Victoria J McCurdy,1 Aime K Johnson,1 Heather Gray-Edwards,1 Brandon L Brunson,1 Ashley N Randle,1 Nancy R Cox,1 Miguel Sena-Esteves,2 Douglas R Martin.1 Auburn University College of Veterinary Medicine, Auburn, AL; 3Gene Therapy Center, University of Massachusetts Medical Center, Worchester, MA The goal of gene therapy is to deliver nucleic acids that encode a therapeutic protein without producing toxicity This requires a delicate balancing act of maximizing protein expression while minimizing vector dosage AAV gene therapy has emerged as a promising treatment for lysosomal storage diseases such as GM2 gangliosidosis (GM2) GM2 is caused by a deficiency of the enzyme Hexosaminidase (Hex), which leads to accumulation of gangliosides in neurons The feline model of this fatal disease has allowed for extensive safety and efficacy testing prior to initiating human clinical trials Safety studies were conducted on six normal cats treated by bilateral thalamic and deep cerebellar nuclei (DCN) injections of AAVrh8 encoding feline Hex There were no clinical signs of toxicity 20 months post-treatment despite achieving extraordinary levels of Hex activity at the cerebral (53-fold normal) and cerebellar (68-fold normal) injection sites Histological analysis of HE sections revealed neurons with hypereosinophilic botryoid inclusions, suggesting high-level expression of the therapeutic protein, which was confirmed by immunostaining with an antibody specific for Hex activity in treated GM2 cats also had hyper-eosinophilic neurons with botryoid inclusions containing high levels of Hex To avoid surgical risks associated with injecting the DCN, three GM2 cats were treated solely by thalamic injections This route proved to significantly prolong survival (p=0.0064) and restored Hex activity to a mean 8.6-fold normal (s.d 1.5) at injection site Thalamic injections proved less efficacious (p=0.0111) than treating both the thalamus and DCN; therefore, alternative routes circumventing the DCN are being explored AAV gene therapy has proven to significantly extend the lifespan of GM2 cats (>4-fold) Despite extremely high levels of Hex activity and protein post-treatment, no evidence of clinical toxicity was observed in normal or GM2 cats 591 Prophylactic and Therapeutic Treatment of Pain in a Varicella- Zoster Virus (VZV) Rodent Model of Post-Herpetic Neuralgia (PHN) Using an HSV Vector Expressing Enkephalin William F Goins,1 Jean-Marc Guedon,2 Mingdi Zhang,1 Joseph C Glorioso,1 Paul R Kinchington.1,2 Microbiology & Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, PA; 2Department of Ophthalmology, University of Pittsburgh School of Medicine, Pittsburgh, PA Varicella-Zoster Virus (VZV) frequently causes pain before and during zoster This pain may become intractable and last longer than 90 days and which is termed Post-Herpetic Neuralgia (PHN) The most distressing and common pain associated with PHN is mechanical allodynia (MA), in which pain results from normal non-painful stimuli such as light touch Current drug therapies (Gabapentin and Amytryptiline) are not efficacious in many PHN patients, providing the impetus for the discovery of novel therapeutics Our group and others have established a VZV-induced rat model of pain in which peripheral injection of VZV induces mechanical hypersensitivity This correlates with transient expression of a subset of VZV genes and maintenance of viral genomes in the dorsal root ganglia (DRG) that innervate the rat hindpaw site of primary infection The sensitivity to mechanical stimuli last 8-10 weeks and reflects long-term allodynia in human PHN patients We evaluated HSV vectors expressing the endogenous opioid enkephalin (ENK) for their ablity to ameliorate VZV-induced nocifensive behaviors Rats were treated with HSV vectors expressing ENK or a GFP-expressing control vector at various doses (104-108 PFU) before or after onset of nocifensive behaviors induced upon VZV inoculation A dose dependent relief of behavioral pain response was observed, with the highest dose animals remaining pain-free for the duration of the experiment (5 weeks) Re-dosing animals with the ENK vector resulted in alleviation of nocifensive behaviors Finally, prophylactic injection of the ENK vector (but not GFP control vector) resulted in a block in the establishment of MA hypersensitivity following VZV injection This study demonstrates that HSV vectors expressing ENK are effective therapeutically and prophylactically in treating pain behaviors in the VZV rat PHN model This suggests the potential of this approach as a viable therapy to treat human PHN patients Nine GM2 cats were treated identically and followed to humane endpoint Cats analyzed to date (n=7) have a mean Hex activity of 24.7-fold normal (s.d 10.5) and 23.1-fold normal (s.d 19.4) at cerebral and cerebellar injection sites, respectively Treatment has significantly prolonged survival to 18.7±7.3 months (>4-fold untreated controls, p=