196 Dosage Thresholds and Vector Serotype Define AAV Targeting of Rod and Cone Photoreceptors in Non Human Primates Molecular Therapy Volume 19, Supplement 1, May 2011 Copyright © The American Society[.]
NEUROLOGIC & OPHTHALMIC GENE & CELL THERAPY I Neurologic & Ophthalmic Gene & Cell Therapy I 194 Long-Term Phenotypic Correction of Feline Lysosomal Storage Disease by Intracranial AAV Gene Therapy Victoria J Jones,1 Allison M Bradbury,1 Misako Hwang,1 Stanley G Leroy,2 Stacy Maitland,2 Aime K Johnson,3 Miguel SenaEsteves,2 Douglas R Martin.1 Scott-Ritchey Res Ctr/Dept Anat, Phys, Pharm, Auburn University College of Vet Med, Auburn, AL; 2Gene Therapy Center, University of Massachusetts Medical School, Worcester, MA; 3Dept Clinical Sciences, Auburn University College of Vet Med, Auburn, AL Deficiency of lysosomal β-galactosidase (βgal) causes storage of GM1 ganglioside, resulting in progressive neurological deterioration and death, often by years of age AAV gene therapy has been extraordinarily successful in the GM1 mouse model, (Mol Ther, 15:30, 2007; PLoS One, 5:e13468, 2010), resulting in enhanced survival and complete clearance of storage in the brains of GM1 mice Because the mouse brain is ∼1000 times smaller and much less complex than the human brain, it is important to test AAV gene therapy in an animal model whose brain size and complexity more closely resemble humans First reported ∼40 years ago, the feline GM1 model presents an unparalleled opportunity to evaluate AAV gene therapy in a non-rodent, ‘large animal’ prior to initiating human clinical trials In the current study, AAV2/1 or AAV2/rh8 vectors expressing a feline βgal cDNA (3.1-12.0e12 g.c total) were injected bilaterally into the thalamus and deep cerebellar nuclei of 2-month old GM1 cats (disease onset ∼3.5 months) In treated brains collected 4-16 weeks post-injection, βgal was distributed throughout the entire anterior-posterior axis of the cerebrum and cerebellum at levels up to times normal Cervical and lumbar spinal cord regions demonstrated βgal activity 0.5 -1 times normal, and filipin staining demonstrated extensive clearance of storage material Little evidence of an inflammatory cellular infiltrate was observed in H&E-stained brain sections, though serum antibody titers to the AAV vectors were pronounced (∼1:65,000) Long-term therapeutic experiments in 2-month old GM1 cats were conducted using the same vector dose and route of delivery Currently, AAV-treated GM1 cats are 16, 14 and 12 months of age, with no evidence of clincal neurological disease (untreated humane endpoint, 7.7 ± 0.8 months, n=9) Treated GM1 cats demonstrate normalization of MRI brain lesions and absence of gait abnormalities typical of untreated cats Other than serum antibody titers, no evidence of vector toxicity has been documented, and no βgal activity was detected in peripheral blood mononuclear cells Further encouraging results come from treatments performed nearer to clinical disease onset (3.0 months of age, weeks prior to disease onset), in which GM1 cats remain neurologically normal at 8.3 months of age These translational studies provide strong support for the initiation of AAV-based clinical trials for human GM1 gangliosidosis 195 Brain Gene Therapy for Metachromatic Leukodystrophy: Optimized Clinical Protocol Caroline Sevin,1,2 Franỗoise Piguet,1 Dolan Sondhi,3 Marie-Anne Colle,4 Thomas Roujeau,1,5 Sylvie Raoul,6 Jack-Yves Deschamps,7 Celine Bouquet,1 Ornella Ahouansou,1 Marie Vanier,8 Philippe Moullier,9 Yan Cherel,4 Neil R Hackett,3 Michel Zerah,1,5 Ronald G Crystal,3 Patrick Aubourg,1,2 Nathalie Cartier.1,2 INSERM U745, Paris, France; 2Pediatric Neurology and Endocrinology, St Vincent de Paul Hospital, Paris, France; 3Weill Medical College of Cornell University, New York; 4UMR INRA 703, Nantes, France; 5Pediatric Neurosurgery, Necker-Enfants Malades Hospital, Paris, France; 6Neurosurgery, CHU Nord, Nantes, France; 7National School of Veterinary Medicine, Nantes, France; 8Inserm U820, Lyon, France; 9Inserm U649, Nantes, France Metachromatic Leukodystrophy (MLD) is a lethal neurodegenerative disease caused by deficiency of Arylsulfatase A (ARSA) The most severe late-infantile form starts around 1-2 years, leading to death within a few years, without available treatment Among potential new therapeutic interventions, brain gene therapy could ensure rapid and sustained delivery of ARSA enzyme in the brain, a prerequisite to arrest the neurodegenerative process in due time We have demonstrated efficiency and safety of intracerebral delivery of adeno-associated-vector serotype (AAV5) encoding human ARSA in MLD mice and non-human primates We recently demonstrated that AAVrh.10 encoding ARSA improves more rapidly MLD mice that AAV5-ARSA and, importantly, allows normalization of sulfatide isoforms that accumulate specifically in oligodendrocytes In a clinical perspective, we optimized in non-human primates the neurosurgical procedure to allow simultaneous infusion of vector at 12 different brain sites in less than 1/2 hours We also developed a brain imaging protocol using MRI to select the injection sites and evaluate the tolerance of the surgical procedure Following the same protocol planed to be used in patients, we demonstrated in non-human primates that intracerebral injection of AAVrh.10/ARSA vector (1.1.10exp11 viral particles per hemisphere in selected areas of the white matter) is well tolerated and results in the diffusion of the vector in 66 to 90% of the injected hemisphere We also documented up to 31% increased activity of ARSA, reaching foreseeable therapeutic levels As compared to our previous results with AAV5/ARSA, the use of a 20-fold lower dose of AAVrh.10 vector injection led to higher amounts of vector and increased level of ARSA activity in the brain Toxicological studies are in progress towards phase I/II tolerance and efficiency clinical trial in late 2011 This trial will enroll five patients with rapidly progressing MLD AAVrh.10/ARSA vector will be administrated to 12 locations in the CNS, guided by brain imaging Safety and efficiency parameters will be evaluated up to years, a period that will be sufficient enough to assess the potential therapeutic efficiency of this brain gene therapy strategy in rapidly progressing forms of MLD 196 Dosage Thresholds and Vector Serotype Define AAV Targeting of Rod and Cone Photoreceptors in Non-Human Primates Luk H Vandenberghe,1 Peter Bell,1 Albert M Maguire,2 Ru Xiao,1 Rebecca Grant,1 Jean Bennett,2 James M Wilson.1 Gene Therapy Program, Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, PA; 2F M Kirby Center for Molecular Ophthalmology, Scheie Eye Institute, University of Pennsylvania, Philadelphia, PA Gene therapy is emerging as a therapeutic modality for treatment of retinal disorders Rod and/or cone photoreceptors are the primary cell types in many inherited retinal degenerations and successful gene therapy for those diseases will therefore require identification S76 Molecular Therapy Volume 19, Supplement 1, May 2011 Copyright © The American Society of Gene & Cell Therapy NEUROLOGIC & OPHTHALMIC GENE & CELL THERAPY I of efficient and safe targeting vectors Previously, we injected nonhuman primate eyes with AAV serotypes via a subretinal route in order to determine relative efficiencies of transduction of the retinal pigment epithelium (RPE) and characterize the histological and immunological consequences of vector administration at various doses Here, we further examined the injected eyes qualitatively and quantitatively for photoreceptor transduction Non-human primates are the only animal models with a macula and therefore we also sought to determine the expression profile in the macula, including the fovea Based on an interesting transduction profile in the murine retina, AAV2, 7, 8, and the engineered capsid isolates rh64R1 and rh8R were subretinally injected into cynomolgus macaque eyes at doses ranging from 10E8 to 10E11 GC per eye of a GFP expressing AAV Eyes were sectioned and analyzed histologically to months following injection Morphometry for transduction efficiency and intensity highlighted substantial differences between vector serotypes AAV8 was the most efficient serotype in transducing photoreceptors at the moderate dose of 10E9 GC At a dose of 10E10 GC however, all serotypes with the exception of AAV2, demonstrated very efficient and roughly equivalent levels of GFP expression in the photoreceptor cell layer Detailed histological analysis of macular regions, including the fovea, and a fourth non-macular site illustrated striking differences in the relative targeting efficiency of cones and rods Whereas all vectors transduced rods quite readily, AAV9 and rh8R emerged as substantially more efficient in cone transduction both within and outside of the fovea At higher doses, the efficiency of cone transduction of the other vectors can be boosted, but nevertheless, the differential permissivity of cones versus rods for each vector remains apparent 197 AAV2/5-Mediated Gene Therapy Synergizes with Bone Marrow Transplantation in the Treatment of Infantile Neuronal Ceroid Lipofuscinosis Shannon L Macauley,1 Marie S Roberts,1 Mark S Sands.1 Internal Medicine, Washington University School of Medicine, Saint Louis, MO Infantile neuronal ceroid lipofuscinosis (INCL) is an inherited, neurodegenerative lysosomal storage disease (LSD) affecting the central nervous system (CNS) during infancy INCL is caused by mutations in the CLN1 gene leading to a deficiency in the lysosomal enzyme, palmitoyl protein thioesterase (PPT1) Pathological changes in the CNS include brain atrophy, cortical thinning, autofluorescent accumulation, neurodegeneration, and glial activation The clinical signs include seizures, blindness, motor dysfunction, mental retardation and premature death by years of age The PPT1/- mouse accurately mimics the human disease PPT1-/- mice have a decreased life span, visual defects, cognitive deficits, epilepsy, and motor dysfunction PPT1-/- brains show similar pathological changes to INCL patients including autofluorescent accumulation, neurodegeneration, glial activation as well as brain atrophy The similarities between PPT1-/- mice and children with INCL make this an excellent tool to investigate therapeutic strategies for the treatment of INCL Currently, gene and stem cell therapies are in clinical trials for the treatment of INCL and late infantile neuronal ceroid lipofuscinosis (LINCL) Although both therapies demonstrated partial improvements on biochemical and histological parameters, the behavioral improvements were modest and no improvement in lifespan was seen Furthermore, another clinical study utilizing bone marrow transplantation (BMT) as a potential therapy for INCL or LINCL proved unsuccessful Thus, given the complexity of INCL and the inadequate therapeutic benefit from any singular therapy, we hypothesized that combining therapeutic modalities must be used to be efficacious In this study, we combined intracranially (IC) delivered AAV2/5 expressing PPT1 with bone marrow transplantation (BMT) At birth, PPT1-/- mice received six injections of AAV2/5-PPT1 Molecular Therapy Volume 19, Supplement 1, May 2011 Copyright © The American Society of Gene & Cell Therapy delivered bilaterally to the anterior cortex, thalamus/hippocampus, and cerebellum The following day the treated PPT1-/- mice were irradiated with a myeloreductive-conditioning regimen (400 rads) followed by BMT via a temporal vein injection of 1x106 bone marrow derived stem cells BMT alone provided no enzyme activity to the brain and did not improve lifespan or motor function, similar to clinical trials Surprisingly, neonatal BMT synergized with IC delivery of AAV2/5-PPT1 to dramatically increase the median lifespan of PPT1-/- mice (untreated PPT1-/- ∼ 8mo, AAV2/5-PPT1only ∼ 13mo, AAV2/5-PPT1/BMT ∼ 17mo) The combination also resulted in sustained improvements in motor function (constant speed and rocking rotorod) On the constant speed rotorod paradigm, the AAV2/5-PPT1+BMT mice performed similar to wildtype mice until 13 months of age and then their performance steadily decreased through 17 months The combination therapy also demonstrated improvements on the rocking test when compared to untreated mice through 11 months of age and then steadily decreased thereafter This combination of CNS-directed gene therapy and BMT demonstrates clinical improvements unparalleled by any previous pre-clinical study or clinical trial done in INCL 198 RNAi Therapy for Spinocerebellar Ataxia Type Megan K Keiser,3 Ryan L Boudreau,1 Beverly L Davidson.1,2,3 Internal Medicine, University of Iowa, Iowa City, IA; 2Neurology, University of Iowa, Iowa City, IA; 3Graduate Program of Neuroscience, University of Iowa, Iowa City, IA Spinocerebellar Ataxia Type (SCA1) is an autosomal dominant late onset neurodegenerative disease SCA1 toxicity is caused by an expanded polyglutamine tract in ataxin-1 The disease affects cerebellar and brainstem neurons, including Purkinje cells Motor symptoms include gait, balance, and motor coordination deficits Currently, there are no curative therapies for SCA1, however, evidence supports that reducing mutant ataxin-1 expression may provide therapeutic benefit RNA interference (RNAi) is a naturally occurring process which mediates gene silencing and is currently being investigated as therapy for dominant diseases such as SCA1 Previous work from our laboratory has shown that RNAi-mediated silencing of a mutant human ataxin-1 transgene improves neuropathological and behavioral phenotypes in a transgenic mouse model of SCA1 The results from this proof-of-concept study highlight the potential of RNAi therapy for treating SCA1 patients Subsequently, we have initiated studies to test this therapeutic strategy in the knock-in mouse model of SCA1, which more closely genocopies human patients We have designed several artificial microRNAs (miRNAs) targeting conserved sequences in both the mouse and human ataxin-1 This approach may facilitate the transition of therapeutic sequences from mouse studies to human We screened the artificial miRNAs in vitro against both transgenic and endogenously expressed ataxin-1 Two therapeutic candidates (siSCA1.1, siSCA1.2) showed effective silencing of ataxin-1 in both human and mouse cell lines siSCA1.1 and siSCA1.2 were placed into adeno-associated viral (AAV) vectors for in vivo delivery Pilot studies showed expression in cerebellar Purkinje cells, knock down of target, and reduction of protein expression At 30 weeks of age (25 weeks after therapy) the SCA1 mutant mice treated with siSCA1.1 trend toward improved motor phenotypes relative to controls Additional tests of therapeutic efficacy are underway This data provides the first evidence of effective RNAi against ataxin-1 in the knock-in model of SCA1 S77 ... I of efficient and safe targeting vectors Previously, we injected nonhuman primate eyes with AAV serotypes via a subretinal route in order to determine relative efficiencies of transduction of. .. macula, including the fovea Based on an interesting transduction profile in the murine retina, AAV2 , 7, 8, and the engineered capsid isolates rh64R1 and rh8R were subretinally injected into cynomolgus... illustrated striking differences in the relative targeting efficiency of cones and rods Whereas all vectors transduced rods quite readily, AAV9 and rh8R emerged as substantially more efficient in cone transduction