586 choroid plexus directed viral gene therapy as a source of alpha n acetyl glucosaminidase enzyme replacement in cerebrospinal fluid for sanfilippo b syndrome

586 Choroid Plexus Directed Viral Gene Therapy as a Source of alpha N Acetyl Glucosaminidase Enzyme Replacement in Cerebrospinal Fluid for Sanfilippo B Syndrome Molecular Therapy Volume 21, Supplement[.]

GENE & CELL THERAPY OF DIABETES, METABOLIC AND GENETIC DISEASES II PCC activity, survive to adulthood, and have significant elevations in propionyl-carnitine, methylcitrate, glycine, alanine, lysine, ammonia, and markers associated with cardiomyopathy similar to those in PA patients This adult model of PA allowed head to head comparison of different gene therapy vectors for their effects on PCCA expression, phenotype, and survival Liver-tropic adenovirus serotype (Ad5) vectors and adeno-associated virus 2/8 (AAV8) vectors were engineered with identical codon-optimized human PCCA cDNAs Ad5 and AAV8 vectors were injected intravenously into adult mice at varied doses and the effects of the two vectors were compared based on the number of PCCA genes that were delivered Both vectors reduced propionyl-carnitine and methylcitrate levels, but Ad5 mediated more rapid increases in PCCA protein and PCC activity in the liver At equal or even 10-fold lower cDNA doses, Ad5 mediated more robust PCCA protein expression in the mice As expected, phenotypic correction was transient with first generation Ad5, whereas AAV8 mediated long-lasting effects Long-term head to head comparisons of cDNA dose by AAV8, AAV-rh10, and helperdependent Ad5 and Ad6 vectors are underway and will be discussed These data suggest that this PA model may be a useful platform for optimizing PA therapeutics 584 Transcription Activator-Like Effector Nuclease-Based Genome Editing for Mucopolysaccharidosis Type I Mark J Osborn,1 Colby G Starker,1 Anthony P DeFeo,1 Daniel F Voytas,1 Bruce R Blazar,1 Jakub Tolar.1 University of Minnesota, Minneapolis, MN Transcription activator like effector nucleases (TALENs) are genome-editing reagents that generate a DNA break at a user-defined site in the genome and hold great promise as a tool for precision gene repair Our goal is to apply TALENs for mucopolysaccharidosis Type IH (MPS IH) Two TALEN candidates, specific for the 392X murine IDUA gene mutation were constructed using the Golden Gate TALEN assembly procedure and tested for their ability to mediate gene editing by homologous recombination of a marker sequence The ability of the TALEN candidates to mediate genomic modification was assessed in murine C1498 cells as well as fibroblasts derived from MPS mice We observed activity consistent with TALEN cutting and DNA repair by the two major repair pathways: non-homologous end joining (NHEJ) and homology directed repair (HDR) We then employed the TALEN in conjunction with an exogenous synthetic donor DNA that contains the corrective base for the 392X mutation that allows for selective outgrowth of corrected cells Following introduction of the TALEN and donor into MPS fibroblasts we were able to document permanent genotypic correction of the 392X mutation by direct sequencing Conclusions: Our results to date show the applicability of genome-editing strategies using engineered nucleases for MPS This provides proof-of-concept for patient-specific gene-editing/ correction in primary human fibroblasts and serves as a platform for translational ex vivo therapies 585 Gene Therapy for Mouse Model of Krabbe Disease Hiroshi Kobayashi,1 Masamichi Ariga,1 Takahiro Fukuda,3 Takeo Iwamoto,4 Sayoko Izuka,1 Yota Shimada,1 Yoshikatsu Eto,2 Toya Ohashi.1,2 Dept of Gene Therapy, Institute of DNA Medicine, Jikei University School of Medicine, Tokyo, Japan; 2Department of Genetic Disease and Genome Science, Jikei University School of Medicine, Tokoy, Japan; 3Department of Neuropathology, Jikei University School of Medicine, Tokyo, Japan; 4Division of Biochemistry, Research Core Facilities, Jikei University School of Medicine, Tokyo, Japan Krabbe disease is genetic neurodegenerative disease, due to genetic deficiency of -galactocerebrosidase (GALC), results in the degeneration of myelin-forming cells (oligodendrocytes and Schwann cells) caused by an accumulation of galactosylsphingosine (psychosine), and the symptoms as the rapid progressive deterioration of the nervous system (especially in infantile form) We tried gene therapy for mouse model of this disease We constructed 2nd generation lentiviral vector, with MCU3 promoter including GALC and GFP expression gene Firstly we checked this vector’s function by transfection for some cell lines as 293A, Twi-2 (fibroblast from tail tip of Krabbe disease model mouse), detected dose dependent enzyme expression And we tried in vivo study with neonatal model mice We injected intravenously for facial vein of neonatal mouse, and detected pathological improvement in accumurative globoid cells in the sciatic nerve, efficient decreasing psychosine accumulation in the brain (p=0.009), long-term expression of viral copy number in some organs including brain using real-time PCR, and improvement in body weight and twitching onset time point We studied the synergy effect of gene therapy and substrate reduction therapy using L-Cycloserine, an irreversible inhibitor of 3-ketodyhydrosphingosine synthase by continuous subcutaneous injection every other day from 5-6 days-old, and detected efficient effect in body weight and life span (p=0.013) For more high viral titer and effective protein synthesis, we also tried gene therapy study using new lentiviral vector including GALC with codon-optimization As a conclusion, recombinant lentiviral vector mediated neonatal gene therapy for Krabbe mouse model should be effective, especially with sustrate reduction thrapy 586 Choroid Plexus-Directed Viral Gene Therapy as a Source of alpha-N-AcetylGlucosaminidase Enzyme Replacement in Cerebrospinal Fluid for Sanfilippo B Syndrome Patricia I Dickson,1 Shih-hsin Kan,1 Steven Q Le,1 Maria Reine Haddad,2 Eun-Young Choi,2 Anthony Donsante,2 Stephen G Kaler.2 Pediatrics, Los Angeles Biomedical Research Institute at HarborUCLA, Torrance, CA; 2Molecular Medicine Program, Eunice Kennedy Shriver National Institute for Child Health and Human Development, National Institutes of Health, Bethesda, MD Mucopolysaccharidosis type IIIB (MPS IIIB; Sanfilippo B) is an inherited lysosomal storage disorder with severe neurological deterioration caused by the deficiency of alphaN-acetyl-glucosaminidase (NAGLU) and is characterized by the accumulation of glycosaminoglycan in the brain Enzyme replacement therapy for MPS IIIB is challenging, due to inadequate mannose 6-phosphorylation (M6P) which limits cellular uptake of recombinantly produced human NAGLU Recently, a modified recombinant human NAGLU fused to the receptor-binding motif of the insulin-like growth factor (rhNAGLU-IGF2) has shown enhanced ability to enter MPS IIIB fibroblasts via IGF2-mediated receptor-mediated endocytosis However, in order to reach the brain as enzyme replacement therapy, rhNAGLU-IGF2 would require delivery by repeated injections into the cerebrospinal fluid (CSF), an S224 Molecular Therapy Volume 21, Supplement 1, May 2013 Copyright © The American Society of Gene & Cell Therapy 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 ... 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... 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... 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;