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74 AAV Gene Therapy with Cholesterol 24 Hydroxylase for Alzheimer Disease In Vivo Consequences on Amyloid and Tau Components of the Pathology Molecular Therapy Volume 20, Supplement 1, May 2012 Copyri[.]
CNS: NEURODEGENERATIVE 72 AAV2/2-CBA-REP1 Vector with WPRE Provides Functional Expression of the Transgene in Choroideremia Mouse Knock-Out and Patient Cells Tanya Tolmachova,1 Oleg E Tolmachov,2 Alun R Barnard,3 Markus Groppe,3 Matthew J During,4 Robert E MacLaren,3,5 Miguel C Seabra.1 Molecular Medicine, NHLI, Imperial College London, United Kingdom; 2Cardiovascular Sciences, NHLI, Imperial College London, United Kingdom; 3The Nuffield Laboratory of Ophthalmology & Oxford Biomedical Research Centre, University of Oxford, United Kingdom; 4Ohio State University Medical Center, Columbus, OH; 5Moorfields Eye Hospital & NIHR Biomedical Research Centre for Ophthalmology, London, United Kingdom Choroideremia (CHM) is an ocular disease due to the loss of function of the CHM/REP1 gene (Xq21.2), which encodes Rab Escort Protein-1 (REP-1) REP1 is important for the lipid modification (prenylation) of Rab GTPases, which are regulators of intracellular vesicular transport First symptoms of CHM, such as night blindness are detected in young teenage patients and are then followed by a slow progressive decline of the vision within next 20-30 years and complete blindness by middle age As a monogenic disorder with a readily available diagnosis and a slow rate of degeneration, CHM is an ideal candidate for addition gene therapy We generated several AAV2-based vectors with CHM/REP1 cDNA and EGFP gene under control of short version of elongation factor-1 alpha promoter (EFS) or CMV-enhanced chicken beta-actin promoter (CBA) Viral vector particles of serotype and were produced The introduction of AAV2/2-CBA-REP1 into HT1080 cells and choroideremia patient fibroblasts resulted in a high level of expression and an increase in prenylation activity, which confirms the functionality of the transgene The insertion of a modified WPRE significantly increased the expression of the transgene The introduction of AAV2/2-CBAREP1 and AAV2/2-CBA-GFP into the mouse retinas by subretinal injection resulted in the expression of the transgene in the RPE and neuroretina Injection of choroideremia mice (Chmnull/wt) with AAV2/2-CBA-REP1 (1-2x10E9 gp, high dose) lead to an increase of ERG responses (both a- and b-wave) in comparison to AAV2/2CBA-GFP injected eyes ERG responses for a low dose viral injection (1-2x10E8 gp) were similar for both AAV2/2-CBA-REP1 and AAV2/2-CBA-GFP Analysis of AAV2/2-CBA-REP1 and sham injected eyes demonstrated an increase for a high dose (1-2x10E9 gp) and a decrease for a low dose (1-2x10E8 gp) in comparison to sham injected eyes Our data demonstrate that the introduction of CHM/ REP1 cDNA leads to the functional expression of the transgene and correction of the prenylation defect in choroideremia cells AAV2/2CBA-REP1 with a modified WPRE is a promising therapeutic agent for choroideremia treatment and has been approved for an ongoing clinical trial (NCT01461213) CNS: Neurodegenerative 73 Allele-Specific Silencing of Mutant Huntingtin in HD Neural Stem Cells and In Vivo Nicole Déglon,1,2,3 Marta Ruiz,2,3 Valérie Drouet,2,3 Diana Zala,4,5 Maxime Feyeux,6 Gwennaelle Auregan,2,3 Raymonde Hassig,2,3 Noelle Dufour,2,3 Anselme Perrier,6 Frédéric Saudou,4,5 Philippe Hantraye.2,3 Department of Clinical Neurosciences (DNC), Laboratory of Cellular and Molecular Neurotherapies (LMCN), Lausanne University Hospital (CHUV), Lausanne, Switzerland; 2Institute of Biomedical Imaging (I2BM) and Molecular Imaging Research Center (MIRCen), CEA, Fontenay-aux-Roses, France; 3URA2210, CNRS, Fontenay-aux-Roses, France; 4Institut Curie, Orsay, France; 5UMR146, CNRS, Orsay, France; 6Unité Mixte de Recherche 861, Institute for Stem Cell Therapy and Exploration of Monogenic Diseases, Association Franỗaise contre les Myopathies, INSERM/Universitộ dEvry-Val-dEssonne, Evry, France The autosomal dominant Huntington’s disease (HD) is a progressive, untreatable, autosomal dominant neurodegenerative disorder resulting from polyglutamine expansion in the huntingtin (Htt) protein Suppression of Htt expression, using RNA interference represents an interesting therapeutic strategy for this incurable pathology However, being able to discriminate wild-type and mutant transcripts might be important to preserve wild-type Htt expression and functions In the present study, we tested the efficacy of two strategies specifically silencing of the mutant htt using four shRNAs either targeting heterozygous single nucleotide polymorphisms SNPs covering the majority of HD patients or the expanded CAG repeats Lentiviral-mediated infection of 293T cells resulted in efficient and selective in vitro silencing of a chimeric mutant Htt reporter system consisting of the sequence of the first 171 amino acids of the mutant human Htt, fused to the Htt exons containing the SNP Furthermore, we showed that the defect in the vesicular transport of BDNF along microtubules was corrected in HD neural stem cells These results were confirmed in a rat model of HD based on the overexpression of the various mutant Htt expressing the SNPs We showed that the shRNAs efficiently degrade Htt mRNA and prevent the apparition of neuropathology when the fully matched mutant Htt was expressed On the contrary, the presence of one mismatch in the targeted mRNA prevented its degradation in almost all cases, leading to the accumulation of Htt aggregates and the appearance of striatal pathology The potential for this allele specific silencing was confirmed by RT-PCR analyses in transgenic BACHD mice and support the therapeutic potential of RNAi for HD 74 AAV Gene Therapy with Cholesterol 24-Hydroxylase for Alzheimer Disease: In Vivo Consequences on Amyloid and Tau Components of the Pathology Benoit Gautier,1 Marie Anne Burlot,1 David Blum,2 Eloïse Hudry,1 Sophie Ayciriex,3 L Troquier,2 N Zommer,2 R Caillerez,2 Vincent Auzeil,3 Jerome Braudeau,1 Olivier Laprevote,3 Laurent Pradier,4 Patrick Aubourg,1 Luc Buée,2 Nathalie Cartier.1 INSERM UMR745, Université René Descartes, Paris, France; INSERM UMR837, Université Lille 2, France; 3Laboratoire de Chimie-Toxicologie Analytique et Cellulaire(C-TAC)EA 4463, Université René Descartes, Paris, France; 4Therapeutic Strategy Unit Aging, Sanofi-Aventis, Chilly-Mazarin, France The development of late onset Alzheimer disease (AD) is closely connected with cholesterol metabolism Cholesterol increases the production of amyloid ß deposits (Aß), a hallmark of AD pathology and is abnormally retained in AD neurons Brain cholesterol is S30 Molecular Therapy Volume 20, Supplement 1, May 2012 Copyright © The American Society of Gene & Cell Therapy CNS: NEURODEGENERATIVE synthesized in situ and excess cholesterol cannot cross the bloodbrain barrier The major exportable form of brain cholesterol is 24S-hydroxy-cholesterol, an oxysterol generated by the key neuronal enzyme cholesterol-24-hydroxylase encoded by CYP46A1 gene We have demonstrated that increasing brain CYP46A1 gene expression through AAV-mediated gene transfer markedly reduces amyloid deposits, Aß40/42 peptides, Aß oligomers, reverses microgliosis and astrocytosis and improves spatial memory defects in APP23 mice before and after the onset of amyloid deposits These data demonstrate that selective overexpression of CYP46A1 in the brain through viral vector delivery of CYP46A1 could find therapeutic applications in Alzheimer disease In a therapeutic perspective we have evaluated the consequence of CYP46A1 overexpression on the Tau pathology that constitutes the second hallmark of AD pathology CYP46A1 overexpression was induced in the cortex and hippocampus of Tau22 mouse model This model exhibits progressive neuron-specific AD like Tau pathology with accumulation of abnormally phosphorylated Tau protein in the hippocampus together with behaviour impairment and electrophysiological alterations AAV5-CYP46A1 was injected in month-old mice Overexpression of CYP46A1 in this model allowed significant improvement of cognitive functions evaluated at months (Morris water maze assay) Mice were sacrificed at months, a time when the pathology is well characterized in this model Neuropathological and biochemical study to further evaluate modulation of Tau pathology and characterize the mechanism of beneficial effect will be presented 75 AAV8-Mediated Expression of VEGF Antagonist Ranibizumab in Macaque Eye: Comparison of Subretinal vs Intravitreal Delivery of Vector Maria P Limberis,1 Ru Xiao,1 Albert M Maguire,2,3 Andras M Komaromy,4 William A Beltran,4 Jean Bennett,2,3 Luk H Vandenberghe,2 James M Wilson.1 Gene Therapy Program, Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia; FM Kirby Center for Molecular Ophthalmology, Scheie Eye Institute, University of Pennsylvania, Philadelphia; 3Center for Cellular and Molecular Therapeutics, Children’s Hospital of Philadelphia; 4Section of Ophthalmology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia Age-related macular degeneration is the leading cause for visual disability among persons over the age of 60 and affects millions of people worldwide Current treatments include vitreal injections of the VEGF antagonists Avastin® or Lucentis® These drugs are expensive, require repeated vitreal injections and while they are initially present at high concentrations, the levels are sub-therapeutic during the long trough period between injections In a nonhuman primate study we evaluated the levels and antiangiogenic activity of AAV8expressed ranibizumab (Lucentis®) Rhesus macaques were injected intravitreally or subretinally with 1011 genome copies of AAV8 vector expressing ranibizumab under the transcriptional control of the CMV promoter No ranibizumab expression was observed in the anterior chamber (AC) fluid of the macaque injected intravitreally (2 eyes/ vector) at any time point examined through to day 217 Following subretinal injection, stable expression of AAV-mediated ranibizumab was observed in the AC fluid that ranged from 615 ng/ml at day 67 to 1312 ng/ml (peak of expression) at day 142, but which reduced to 82 ng/ml at day 217 To pharmacologically regulate ranibizumab expression in the retina, we injected the AAV8-ARGENTTM vector subretinally followed by an intravenous injection of rapamycin to induce expression Levels of ranibizumab were undetectable prior to induction and almost 200 ng/ml within 14 days of induction The antiangiogenic activity of ranibizumab produced in the AC fluid by the constitutive or the ARGENTTM vector was assayed in the HUVEC Molecular Therapy Volume 20, Supplement 1, May 2012 Copyright © The American Society of Gene & Cell Therapy in vitro angiogenesis model AAV8-expressed ranibizumab inhibited VEGF-induced proliferation by ∼75% In summary, an AAV8 vector can express clinically relevant levels of the VEGF antagonist ranibizumab in the macaque eye, the expression of which can be regulated by a safe pharmacological approach 76 Identifying Motor Neuron Transduction Efficiencies That Are Efficacious in SMA Mice and Achievable by Intrathecal Delivery in a Large Animal Model Jie Bu,1 Amy M Richards,1 Christopher M Treleaven,1 Catherine R O’Riordan,1 Thais Federici,2 Nicholas M Boulis,2 Seng H Cheng,1 Lamya S Shihabuddin,1 Marco A Passini.1 Neuroscience, Genzyme Corporation, Framingham, MA; Neurosurgery, Emory University, Atlanta, GA Spinal muscular atrophy (SMA) is a pediatric neuromuscular disease caused by a deficiency in SMN protein due to mutations in SMN1 A decrease in SMN levels results in motor neuron cell death in the spinal cord that leads to weakness and wasting of the muscles responsible for locomotion, swallowing, breathing and coughing We previously reported that CNS-targeted AAV-hSMN therapy is highly efficacious in SMA mice (J Clin Invest 120:1253-1264, 2010) Importantly, the demonstration that both the spinal cord and muscle can be corrected with CNS delivery allows for the development of a single route of administration to treat SMA in the clinic Intrathecal (IT) delivery is an attractive method because the catheter does not pierce the pia of the spinal cord, and the smaller doses and decreased possibility of an immune response against capsid proteins makes this delivery method more amenable for clinical translation than systemic injections The goals of the current study is to 1) determine the minimum percentage of spinal cord motor neurons that needs to be transduced for efficacy in SMA mice, and 2) determine if this motor neuron transduction rate can be achieved by IT delivery in large animal models We hypothesize that AAV vector dose correlates with motor neuron transduction efficiencies to impact survival in SMA mice Thus, doses of 5e10, 1e10, and 1e9 genome copies (gc) of scAAV9-hSMN were injected into the CNS of SMA mice Treatment resulted in median lifespans of 153d (+800% increase compared to saline controls, p