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[.]
Trang 1Molecular Therapy Volume 20, Supplement 1, May 2012 Copyright © The American Society of Gene & Cell Therapy
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CNS: NEURODEGENERATIVE
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
1 Molecular Medicine, NHLI, Imperial College London,
United Kingdom; 2 Cardiovascular Sciences, NHLI, Imperial
College London, United Kingdom; 3 The Nuffi eld Laboratory of
Ophthalmology & Oxford Biomedical Research Centre, University
of Oxford, United Kingdom; 4 Ohio State University Medical
Center, Columbus, OH; 5 Moorfi elds 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 modifi cation
(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 2 and 5 were produced The introduction of
AAV2/2-CBA-REP1 into HT1080 cells and choroideremia patient
fi broblasts resulted in a high level of expression and an increase
in prenylation activity, which confi rms the functionality of the
transgene The insertion of a modifi ed WPRE signifi cantly increased
the expression of the transgene The introduction of
AAV2/2-CBA-REP1 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
AAV2/2-CBA-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
AAV2/2-CBA-REP1 with a modifi ed WPRE is a promising therapeutic agent
for choroideremia treatment and has been approved for an ongoing
clinical trial (NCT01461213)
CNS: Neurodegenerative
73 Allele-Specifi c 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
1 Department of Clinical Neurosciences (DNC), Laboratory of Cellular and Molecular Neurotherapies (LMCN), Lausanne University Hospital (CHUV), Lausanne, Switzerland; 2 Institute
of Biomedical Imaging (I2BM) and Molecular Imaging Research Center (MIRCen), CEA, Fontenay-aux-Roses, France; 3 URA2210, CNRS, Fontenay-aux-Roses, France; 4 Institut Curie, Orsay, France; 5 UMR146, CNRS, Orsay, France; 6 Unité Mixte de Recherche 861, Institute for Stem Cell Therapy and Exploration
of Monogenic Diseases, Association Française contre les Myopathies, INSERM/Université d’Evry-Val-d’Essonne, 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 effi cacy of two strategies specifi cally 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 effi cient and selective in vitro silencing of a chimeric mutant Htt reporter system consisting of the sequence of the fi rst 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 confi rmed in a rat model of HD based on the overexpression of the various mutant Htt expressing the SNPs We showed that the shRNAs effi ciently 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 specifi c silencing was confi rmed by RT-PCR analyses in transgenic BACHD mice and support the therapeutic potential of RNAi for HD
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 Elọse 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
1 INSERM UMR745, Université René Descartes, Paris, France;
2 INSERM UMR837, Université Lille 2, France; 3 Laboratoire de Chimie-Toxicologie Analytique et Cellulaire(C-TAC)EA 4463, Université René Descartes, Paris, France; 4 Therapeutic 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
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CNS: NEURODEGENERATIVE
synthesized in situ and excess cholesterol cannot cross the
blood-brain barrier The major exportable form of blood-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 fi nd 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-specifi c 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 3 month-old mice Overexpression of CYP46A1 in this model
allowed signifi cant improvement of cognitive functions evaluated
at 9 months (Morris water maze assay) Mice were sacrifi ced at
9 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
benefi cial effect will be presented
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
1 Gene Therapy Program, Department of Pathology and
Laboratory Medicine, University of Pennsylvania, Philadelphia;
2 FM Kirby Center for Molecular Ophthalmology, Scheie Eye
Institute, University of Pennsylvania, Philadelphia; 3 Center for
Cellular and Molecular Therapeutics, Children’s Hospital of
Philadelphia; 4 Section 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
AAV8-expressed 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) fl uid 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 fl uid 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 fl uid by
the constitutive or the ARGENTTM vector was assayed in the HUVEC
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
Effi ciencies That Are Effi cacious 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
1 Neuroscience, Genzyme Corporation, Framingham, MA;
2 Neurosurgery, Emory University, Atlanta, GA.
Spinal muscular atrophy (SMA) is a pediatric neuromuscular disease caused by a defi ciency 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 effi cacious 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 effi cacy 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 effi ciencies 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<0.0001), 70d (+312%, p<0.0001), and 18d (+6%, p=0.1329), respectively Grip strength and righting refl ex tests in mice administered the two highest doses showed signifi cant and sustained improvements in muscle strength and coordination at 14d and 175d Analysis of the spinal cord at 14d in mice treated with 5e10, 1e10, or 1e9 gc resulted in motor neuron transduction effi ciencies
of 30-60%, 10-30%, and <5%, respectively These data indicate that transducing a minimum of 10-30% motor neurons is suffi cient
to improve function and survival in SMA mice, thus establishing a benchmark criteria for success in subsequent bio-delivery experiments
in large animal models To this end, we performed an IT study in juvenile farm pigs to determine the feasibility of widespread gene delivery in a model whose spinal cord size and anatomy is similar
to that of humans A comprehensive analysis with scAAV9-eGFP (dose = 3e12 gc) showed robust vector expression in the ventral horn of the cervical, thoracic, and lumbar regions at 30 days post-IT-injection Importantly, double IHC with eGFP and ChAT produced
a motor neuron transduction effi ciency of 10-60% in the majority
of spinal cord segments analyzed This data confi rms that a motor neuron transduction pattern predicted to be effi cacious is achievable using a clinically viable delivery method, and justifi es the continual development of gene therapy for SMA