502 Optimization of Intrathecal Delivery of AAV for Targeting the Spinal Compartment Molecular Therapy Volume 23, Supplement 1, May 2015 Copyright © The American Society of Gene & Cell Therapy S200 NE[.]
NEUROLOGIC DISEASES (INCLUDING OPHTHALMIC AND AUDITORY DISEASES) II 500 Triple AAV Vectors Expand AAV Cargo Capacity in the Retina Andrea Maddalena,1 Pasqualina Colella,1 Ivana Trapani,1 Renato Minopoli,1 Alberto Auricchio.1 Telethon Institute of Genetics and Medicine (TIGEM), Pozzuoli, Naples, Italy We have recently shown that dual AAV vectors expand AAV cargo capacity in the retina up to 9kb thus allowing their application to the therapy of inherited blinding conditions like Stargardt disease or Usher syndrome type IB (USHIB) However, for several other inherited blinding conditions the size of the coding sequence to be transferred would not fit into dual AAV vectors and would require a triple AAV vector system We have generated triple AAV vectors encoding a reporter EGFP-DsRed fusion protein under the transcriptional control of the ubiquitous CMV promoter The expression cassette was split in three parts and packaged in three distinct AAV vectors In order to favour the directional concatemerization upon co-infection, highly recombinogenic sequences were added to the constructs which would then be eliminated from the mature transcript by the inclusion of flanking splicing signals (Figure 1) Triple AAV 2/2 vectors were used to infect HEK293 cells in vitro and AAV 2/8 vectors in vivo by subretinal administration in C57Bl/6 mice Direct fluorescence analysis showed EGFP and DsRed colocalization in HEK293 cells; full length protein expression was confirmed by Western Blot analysis of HEK293 cell lysates Fundoscopy analysis of C57Bl/6 mice up to months after subretinal delivery shows signal compatible with EGFP-DsRed expression Western-blot analysis of retinal lysates supports the production of the full length protein Our results suggest that AAV DNA cargo capacity can be increased up to 12kb in the retina by triple AAV vectors This bodes well for gene therapy of retinal diseases which require a high DNA cargo capacity non-invasive determination of spatial distribution using MicroPET imaging The production of AAVrh.10CLN2 under GMP conditions met endotoxin, mycoplasma, sterility and transgene expression release criteria Purified AAVrh.10CLN2 was concentrated to 1013 gene copies/ml Labeling with Na124I was carried out at 2-5 0C under mild oxidizing conditions in pH 7.5 buffer Following radiolabeling, the product mixture was purified using an anion exchange cartridge and centrifugal filtration Purified I124-AAVrh.10CLN2 was formulated in a pH 7.4 PBS buffer The sterile formulation was injected (2 ml at 2.5 µCi/ml) intraparenchymally to the striatum in the murine brain and imaged on a Siemens Inveon MicroPET scanner Thirty minute PET scans were acquired for each mouse (n=3) For the control group, we performed the same procedure using free Na 124I alone (n=3) The radiolabeling efficiency of AAV was in the range of 12-14% PET/ CT imaging clearly demonstrated the spatial distribution of vector over a ten day period, with minimal 124I uptake in the unblocked thyroid In contrast, free iodide was rapidly cleared from the brain within days (Figure 1) In conclusion, adeno-associated virus was successfully labeled with 124I and its distribution in the mouse brain was observed This radiolabeling approach has the potential for wide application in gene therapy trials 502 Optimization of Intrathecal Delivery of AAV for Targeting the Spinal Compartment 501 Radioiodinated Adeno-Associated Virus: A Promising New Approach for Monitoring Gene Therapy Paresh J Kothari,1 Bishnu P De,2 Bin He,1 Conor Foley,1 Alvin Chen,2 Maria J Chiuchiolo,2 Dolan Sondhi,2 Stephen Kaminsky,2 John Babich,1 Shankar Vallabajosula,1 Ronald G Crystal,2 Douglas Ballon.1 Department of Radiology, Weill cornell Medical College, New York, NY; 2Depatment of Genetic Medicine, Weill Cornell Medical college, New York, NY Late infantile neuronal ceroid lipofuscinosis (LINCL) is a form of Batten disease that is caused by mutations in the CLN2 gene These defects cause widespread neurodegeneration resulting in death by the age of 10-12 years One treatment for LINCL that has shown promise in animal and clinical studies is gene therapy using adeno-associated virus (AAV) as a vehicle to deliver the CLN2 gene throughout the brain This is currently accomplished by direct infusion, but there is no way to measure the spatial distribution of administered vector Iodine-124 labeling of the viral capsid may offer a means for S200 Adrian P Kells,1 Martin Goulet,1 Justin Aubin,1 Shipeng Yuan,1 David Dismuke,1 Randall P Reed,2 Qin Su,3 Guangping Gao,3 Dinah W.Y Sah,1 Gregory R Stewart.1 Voyager Therapeutics Inc., Cambridge, MA; 2Northern Biomedical Research, Spring Lake, MI; 3Gene Therapy Center, University of Massachusetts Medical School, Worcester, MA Adeno-associated viral (AAV) vectors are a platform of great potential for therapeutic gene delivery One of the major challenges regarding AAV gene therapy is to deliver the transgene of interest to target cells at levels that result in expression that is both safe and effective For diseases of the central nervous system (CNS) such as amyotrophic lateral sclerosis (ALS) and Friedreich’s ataxia, it is important to identify a dosing paradigm that provides a relatively homogenous distribution of gene transfer along the rostral-caudal axis of the spinal column in the CNS and is translatable Intrathecal (IT) administration is a delivery approach that has shown promise for providing such distribution AAV dosing via the IT route has been reported in large mammals to provide greater CNS distribution, less exposure to peripheral organs and tissues, and reduced impact of immune responses than systemic dosing However, to-date, IT dosing of AAV in large mammals has been investigated primarily by lumbar bolus administration, with a few studies assessing administration at Molecular Therapy Volume 23, Supplement 1, May 2015 Copyright © The American Society of Gene & Cell Therapy Musculo-Skeletal Diseases more rostral sites either alone or in combination with a lumbar site Parameters that are likely to have a major impact on CNS distribution such as volume, rate and duration of infusion have not been reported previously for IT dosing of AAV Here, we studied the effects of these parameters as well as site of infusion on distribution of transgene expression in the non-human primate CNS, using AAVrh10 to package a vector genome (vg) containing the human frataxin gene driven by the chicken β-actin promoter Four weeks after dosing, frataxin (FXN) expression was assessed in spinal cord and dorsal root ganglia (DRG) at multiple rostral-caudal levels, as well as in brain, cerebellum, and peripheral organs such as liver, spleen and heart Quantitative assessments included measurements of vector genome copy number and mRNA and protein expression levels Our results showed that the distribution of transgene expression in the spinal cord, DRG, cerebellum and brain were significantly impacted by the site(s) of IT delivery (i.e cervical vs lumbar), and volume, rate and duration of infusion As expected, there was transduction of peripheral organs, indicating systemic exposure of the AAV vector following IT delivery In addition, FXN expression across different cell types is being studied by histological methods These results will not only guide the optimization of IT delivery of AAV gene therapy for diseases such as ALS and Friedreich’s ataxia, but also provide useful information for IT dosing for other CNS disorders controls (N=6); affected dogs given saline only (N=4) or affected dogs given AAV8-MTM1 at the following dosages: (5E12 vg/kg, N=3), (2.5E13 vg/kg, N=3) or (8E13 vg/kg, N=3) AAV8-MTM1 was administered at 10 weeks of age and dogs were followed for months Blinded analyses included limb strength, gait, respiratory function, neurological assessment, histology, vector biodistribution, transgene expression and immune response over the nine month study Pathology characteristic of XLMTM was moderately improved in the 5E12 vg/kg dosed group and was dramatically improved in the animals receiving (2.5E13 vg/kg) or (8E13 vg/kg) doses of AAV Musculo-Skeletal Diseases 503 Minimally Effective Dose of Systemic AAV8-MTM1 Needed To Prolong Survival and Correct Severe Muscle Pathology in a Canine Model of X-Linked Myotubular Myopathy David L Mack,1,2 Karine Poulard,9 Melissa Goddard,3 Jessica M Snyder,4 Robert W Grange,5 Jon Doering,5 Stefan Czerniecki,1,2 Jennifer L Strande,6 Virginie Latournerie,9 Philippe Veron,9,10,11 Hui Meng,7 Lin Yang,8 Fujun Liu,8 Christine Le Bec,9 Laurine Buscara,9 Samia Martin,9 Philippe Moullier,9,10,11 Michael O’Callaghan,12 Federico Mingozzi,9,14 Alan H Beggs,13 Michael W Lawlor,7 Anna Buj-Bello,9 Fulvio Mavilio,9 Martin K Childers.1,2 Rehabilitation Medicine, University of Washington, Seattle, WA; Institute for Stem Cell and Regenerative Medicine, Seattle, WA; Physiology and Pharmacology, Wake Forest University, WinstonSalem, NC; 4Comparative Medicine, University of Washington, Seattle, WA; 5Human Nutrition, Foods and Exercise, Virginia Tech, Blacksburg, VA; 6Medicine; Cell Biology, Neurobiology and Anatomy; Cardiovascular Center, Medical College of Wisconsin, Milwaukee; 7Division of Pediatric Pathology, Department of Pathology and Laboratory Medicine, Medical College of Wisconsin, Milwaukee; 8Biomedical Engineering, University of Florida, Gainesville, FL; 9Genethon, Evry, France; 10Laboratoire de Therapie Genique, INSERM U649, Nantes, France; 11Molecular Genetics and Microbiology, University of Florida, Gainesville, FL; 12 Audentes Therapeutics, San Francisco, CA; 13The Manton Center for Orphan Disease Research, Harvard Medical School, Boston, MA; 14University Pierre and Marie Curie, Paris, France Mutations in the myotubularin gene (MTM1) result in X-linked myotubular myopathy (XLMTM), a fatal pediatric disease characterized by small centrally nucleated myofibers Patients typically present with severe hypotonia and respiratory failure We previously reported that administration of adeno-associated virus serotype vector (AAV8) expressing myotubularin under the musclespecific desmin promoter delivered intravenously (i.v.) in Mtm1-KO mice and by regional hind limb perfusion in myotubularin-deficient dogs prolonged life and restored muscle function Here we report the results of an i.v dose escalation study testing the efficacy of AAV8-MTM1 in XLMTM dogs Five groups were studied: Normal Molecular Therapy Volume 23, Supplement 1, May 2015 Copyright © The American Society of Gene & Cell Therapy 504 Targeted Genome Engineering of Induced Pluripotent Stem Cells To Produce Auto-Regulated Inflammation Resistance for Musculoskeletal Regenerative Medicine Jonathan M Brunger,1 Ananya Zutshi,1 Vincent P Willard,1 Charles A Gersbach,1 Farshid Guilak.1 Orthopaedic Surgery and Biomedical Engineering, Duke University, Durham, NC Chronic inflammatory diseases such as arthritis are characterized by aberrant and dysregulated responses to cytokines such as tumor necrosis factor α (TNFα) While exogenous anti-cytokine therapies have been shown to diminish this inflammatory response, they are often used at high, unregulated doses that can induce significant side effects Here, we propose a novel regenerative medicine approach to treating such chronic inflammatory diseases by generating customdesigned cells that can execute real-time, user-defined responses to environmental cues, including pro-inflammatory cytokines We designed and deployed gene editing nucleases based on the CRISPR/ Cas9 system to create stem cells with the ability to antagonize TNFαmediated inflammation in an auto-regulated, feedback-controlled manner for musculoskeletal regenerative medicine applications Specifically, the cytokine-responsive Ccl2 gene was reprogrammed by nuclease-mediated integration of the gene encoding soluble TNFα receptor I (sTNFRI), a TNFα antagonist, immediately downstream S201 ... previously for IT dosing of AAV Here, we studied the effects of these parameters as well as site of infusion on distribution of transgene expression in the non-human primate CNS, using AAVrh10 to... by the site(s) of IT delivery (i.e cervical vs lumbar), and volume, rate and duration of infusion As expected, there was transduction of peripheral organs, indicating systemic exposure of the AAV. .. IT delivery In addition, FXN expression across different cell types is being studied by histological methods These results will not only guide the optimization of IT delivery of AAV gene therapy