124 AAV Mediated Antisense Oligonucleotide Based Therapy for CEP290 Associated LCA Molecular Therapy Volume 22, Supplement 1, May 2014 Copyright © The American Society of Gene & Cell Therapy S46 SENSO[.]
SENSORY (OPHTHALMIC AND AUDITORY) DISEASES for gene therapy treatment is larger than the current packaging capacity of wildtype AAV Mutations in crb1, initially identified in Drosophila, are associated with Leber congenital amaurosis and multiple forms of autosomal recessive retinitis pigmentosa The crb1 protein localizes to the subapical region in the outerlimiting membrane where photoreceptors contact Müller glia and is necessary for cell-cell adhesion The therapeutic strategy to treat this disease is the delivery of a functional copy of crb1 to the Müller glia using a viral vector, such as AAV Our group has previously developed an AAV vector, ShH10, capable of specifically targeting Müller glia allowing for cell-specific delivery of genetic cargo Delivering crb1 with ShH10 suffers from the same problem as wildtype AAV serotypes in that the size of the necessary transgene is too large for efficient viral packaging We are overcoming this limitation using two distinct approaches in order to develop a successful gene therapy for patients suffering from retinal degenerations caused by mutations in crb1 The first approach is to use small synthetic CMV promoters to drive expression of crb1, while still remaining within the size constraint of wildtype AAV By utilizing 113 bp and 323 bp synthetic CMV-based promoters, the necessary transgene is between 4.7 - kb Here, we demonstrate that both minimal promoters are capable of driving expression in the mouse retina when delivered via ShH10 The second approach is to utilize directed evolution of the viral capsid to increase the carrying capacity of AAV This would allow for the delivery of the oversized crb1 transgene to Müller glia We have developed a methodology for screening a large library of AAV variants for the capability of carrying larger genetic cargo By increasing the carrying capacity of AAV, the transgene size constraint of wildtype AAV will be obviated not only for crb1 but many other transgenes currently too large for wildtype AAV delivery 122 A Long-Term Study of AAV Vector-Mediated Gene Therapy for X-linked Retinitis Pigmentosa (XLRP) Due to RP2 Mutation Suddhasil Mookherjee,1 Peter Colosi,1 Suja Hiriyanna,1 Kayleigh Kaneshiro,1 Linjing Li,2 Haohua Qian,1 Tiansen Li,1 Hemant Khanna,2 Anand Swaroop,1 Zhijian Wu.1 National Eye Institute, National Institute of Health, Bethesda, MD; 2Department of Ophthalmology, University of Massachusetts Medical School, Worcester, MA Retinitis pigmentosa (RP) is a degenerating eye disorder associated with early night blindness, progressive loss of peripheral vision and eventual loss of central vision The mode of inheritance of the disease can be autosomal dominant, recessive or X-linked Mutations in RP2 gene account for a total of 10-20% of X-linked RP cases No treatment is available for the disease yet In the present study, we designed and generated a self-complementary AAV8 vector containing human RP2 cDNA with a rhodopsin kinase promoter, a CMV/βGlobin intron and a human β-globin poly A tail, and tested its efficacy in complementing the Rp2 function in an Rp2 knockout mouse model The mice were treated unilaterally with a single subretinal injection of the vector at 4-6 weeks of age The fellow eyes were injected with saline as a control Three doses ranging from 1e8 to 1e9 vector genomes (vg) per eye were tested Expression of human RP2 protein was detected in the photoreceptor layer of vector-treated retina Western blot demonstrated the expected molecular weight of the expressed RP2 protein A significantly higher photopic ERG response was revealed in vector-treated eyes compared to the salineinjected eyes between and 18 months of age for all vector doses The treated eyes also showed higher visual acuity as evidenced by the results of optokinetic test Consistent with these observations, immunohistochemical analysis indicated higher cone density in the vector-treated eyes at the final 18 month time point Due to the slow rod degeneration in this mouse model, no significant difference was observed between the vector-treated and the control eyes in either S46 the scotopic ERG amplitude or the outer nuclear layer thickness at the dose of 1e8 vg/eye A decline in the scotopic ERG was observed in 1e9 and 3e8 vg/eye groups, indicating the vector toxicity to rods at these higher doses Our study demonstrates that an AAV8- RP2 vector preserves cone function and promotes cone survival in an Rp2 knockout mouse model of retinitis pigmentosa As a potential clinical candidate for the disease, a detailed dose-efficacy profile of the vector is being investigated 123 Efficient Gene Delivery To the ConeEnriched Pig Retina By Dual AAV Vectors Pasqualina Colella,1 Ivana Trapani,1 Giulia Cesi,1 Andrea Sommella,1 Anna Manfredi,1 Agostina Puppo,1 Carolina Iodice,1 Settimio Rossi,2 Francesca Simonelli,2 Massimo Giunti,3 Maria Laura Bacci,3 Alberto Auricchio.1,4 Telethon Institute of Genetics and Medicine (TIGEM), Naples, Italy; 2Department of Ophthalmology, Second University of Naples, Naples, Italy; 3Department of Veterinary Morphophysiology and Animal Production, University of Bologna, Bologna, Italy; 4Medical Genetics, Department of Translational Medicine, Federico II University, Naples, Italy Gene therapy with adeno-associated viral (AAV) vectors is limited by AAV cargo capacity that prevents their application to those inherited retinal diseases (IRDs), as Stargardt’s disease (STGD) or Usher syndrome type IB (USH1B) that are due to mutations in genes larger than Kb Trans-splicing or hybrid dual AAV vectors have been successfully exploited to reconstitute large gene expression in the mouse retina Here we tested them in the large cone-enriched pig retina, which closely mimics human retina We found that dual AAV trans-splicing and hybrid vectors transduce pig photoreceptors, the major cell targets for treatment of IRDs, to levels that were about 2-3 folds lower than those obtained with a single AAV vector of normal size This efficiency is significantly higher than in mice and potentially due to the high levels of dual AAV co-transduction we observe in pigs We also show that subretinal delivery in pigs of dual AAV trans-splicing and hybrid vectors successfully reconstitute, albeit at variable levels, the expression of the large genes ABCA4 and MYO7A mutated in STGD and USH1B, respectively Our data support the potential of dual AAV vectors for large gene reconstitution in the cone-enriched pig retina that is a relevant pre-clinical model 124 AAV-Mediated Antisense OligonucleotideBased Therapy for CEP290-Associated LCA Alejandro Garanto,1 Lonneke E M Duijkers,1 Ru Xiao,2 Luk H Vandenberghe,2 Rob W J Collin.1 Human Genetics, Radboud University Medical Centre, Nijmegen, Netherlands; 2Ophthalmology, Schepens Eye Research Institute, Boston Leber congenital amaurosis (LCA) is a severe form of retinal degeneration that can be caused by mutations in more than 20 different genes The most recurrent causative mutation is an intronic change in CEP290 (c.2991+1655A>G), that results in the insertion of a cryptic exon into CEP290 mRNA Antisense oligonucleotides (AONs) are small RNA molecules able to redirect pre-mRNA splicing of specific targets Recently, we have shown promising results employing AONbased therapy in cultured lymphoblastoid and fibroblast cells of LCA patients homozygously carrying the intronic CEP290 mutation Correct splicing, ciliation and cilium length were almost completely rescued, and CEP290 protein levels significantly increased after AON treatment The aim of this work is to further develop this therapeutic strategy, by employing viral vectors that express these AONs Different AONs were cloned into AAV vectors and their efficacy was assessed by co-transfecting the different AONs together with a mini-gene containing the intronic mutation RT-PCR analysis revealed Molecular Therapy Volume 22, Supplement 1, May 2014 Copyright © The American Society of Gene & Cell Therapy SENSORY (OPHTHALMIC AND AUDITORY) DISEASES two highly effective AON-containing AAV-vectors that were able to redirect normal CEP290 pre-mRNA splicing In addition, we have assessed the transduction efficacy of a panel of differnet AAV serotypes for transducing cultured fibroblast cells Currently, we are generating AON-containing AAVs of the most potent serotype, and assessing their therapeutic efficacy by RT-PCR, immunodetection of CEP290 protein levels, and measuring ciliation and cilium length in patient-derived fibroblast cells Herewith, we demonstrate the therapeutic potential of AAV-based delivery of AONs to treat CEP290-associated LCA 125 Hybrid VEGF/PDGF Soluble Receptors for Inhibition of Ocular Neovascularization Peter Pechan,1 Jeffery Ardinger,1 Hillard Rubin,1 Michael Lukason,1 Elizabeth Barry,1 Samuel C Wadsworth,1 Abraham Scaria.1 Gene Therapy, Sanofi-Genzyme R&D Center, Framingham, MA Pathological neovascularization (NV) is a key component of the wet form of age-related macular degeneration (wet AMD or NVAMD) There are several reports of preclinical studies showing that anti-angiogenesis may be an efficient strategy for treating NV-AMD in animal models Vascular endothelial growth factor A (VEGF) is one of the main inducers of ocular NV and several clinical trials have now taken the approach of neutralizing VEGF to treat patients with NV-AMD and diabetic macular edema (DME) Wet age-related macular degeneration (wet AMD) is the leading cause of central vision loss in individuals 65 years of age and older Here, we test whether combined antagonism of both VEGF and platelet-derived growth factor-B (PDGF) represents a more efficient strategy for treating wet AMD Starting with the full-length PDGF receptor β (PDGFRβ) ectodomain (ECD), we have engineered several truncated and hybrid PDGF and VEGF-binding soluble receptors The biological activity of these constructs was determined using the HUVECs proliferation assay, VEGF and PDGF binding assays Of the different truncated soluble PDGFRβ constructs generated, we determined that the first three PDGFRβ ECD domains are necessary for efficient binding to PDGF The most effective dual PDGF and VEGF binder was construct Hybrid #4, which contained the first three PDGFRβ ECD domains fused to domain of Flt-1 via a peptide linker To test its efficacy in vivo, a recombinant AAV2 vector encoding Hybrid #4 was generated and injected intravitreally into a mouse model subjected to laser-induced pathological NV Hybrid #4 construct was effective in inhibiting laser-induced ocular NV, illustrating the potential of targeting PDGF and VEGF to treat pathological NV (e.g., wet AMD) 126 Retinoschisin Gene Therapy in Photoreceptors, Muller Glia, or All Retinal Cells in the Mouse Model of X-Linked Retinoschisis Leah C Byrne,1 Bilge E Ozturk,1 Trevor Lee,1 Cecile Fortuny,1 Meike Visel,1 Deniz Dalkara,1 David V Schaffer,2 John G Flannery.1 Helen Wills Neuroscience Institute, University of California, Berkeley, Berkeley, CA; 2Department of Chemical and Biomolecular Engineering, University of California, Berkeley, Berkeley, CA However, photoreceptors, not glia, normally secrete retinoschisin We compared the efficacy of rescue mediated by retinoschisin secretion from these specific subtypes of retinal cells in the Rs1h/- mouse model of retinoschisis Our results indicate that all three vectors deliver the RS1 gene, and that several cell types can secrete retinoschisin, leading to diffusion of the protein across the retina The greatest long-term rescue was observed when photoreceptors produce retinoschisin Similar rescue was observed with photoreceptorspecific or generalized expression, though photoreceptor secretion may contribute to rescue in the latter case These results collectively point to the importance of cell targeting and appropriate vector choice in the success of retinal gene therapies 127 Efficient Protection of Retina By TyrosineMutated Self-Complementary AAV2 Vector Encoding BDNF in a Rat Retinal Ischemic Injury Model Tsutomu Igarashi,1,2 Koichi Miyake,2 Maika Kobayashi,1,2 Kazuhisa Takahashi,1,2 Noriko Miyake,2 Osamu Iijima,2 Kenji Nakamoto,1 Yukihiko Hirai,2 Takashi Shimada,2 Hiroshi Takahashi.1 Ophthalmology, Nippon Medical School, Tokyo, Japan; Department of Biochemistry and Molecular Biology, Division of Gene Therapy Research Center for Advanced Medical Technology, Nippon Medical School, Tokyo, Japan Ocular disorders are promising targets for gene therapy, which is becoming a well-established field Glaucoma and retinal artery occlusion are caused by an injury of retinal ganglion cell (RGC) Recently, it was reported that brain-derived neurotrophic factor (BDNF) have a function of rodent RGC neuroprotection In this study, we examined the feasibility of self-complementary adeno-associated virus (scAAV) vector mediated gene therapy for glaucoma and retinal artery occlusion using rat retinal ischemic injury model To transduce with high efficiency, we generated type mutant (triple Y-F) scAAV vector expressing BDNF (scAAV2-BDNF) and intravitreal injection was performed We used type mutant scAAV vector expressing GFP (scAAV2-GFP) as a control Two weeks after injection, retinal ischemia was induced in rats by raising intraocular pressure (IOP) to 110 mm Hg for 60 mins The neuroprotective effects of BDNF were evaluated by determining the preservation of the inner retina thickness and one week after reperfusion In addition, electroretinograms (ERGs) were performed to determine the functionality of the retina Significant improvement of the inner retinal thickness was observed in scAAV2-BDNF treated eyes compared to scAAV-GFP treated control eyes (No treatment; 78 ± 3.6 μm, Control; 42 ± 10.3 μm, scAAV2BDNF; 77 ± 8.1 μm.) Moreover, ERGs showed that retinal function also rescued by scAAV2-BDNF treatment (b-wave: No treatment; 975 ± 191 μV, Control; 396 ± 231 μV, scAAV-BDNF; 882 ± 155 μV) Thus, triple mutant scAAV type vector mediated BDNF extremely protected rat retina from ischemia-reperfusion injury These results suggested that in vivo gene therapy using BDNF appears to be a feasible approach to the clinical management of neuroprotection in conditions such as glaucoma and retinal artery occlusion X-linked retinoschisis, a disease characterized by splitting of the retina, is caused by mutations in the retinoschisin gene, which encodes a secreted cell adhesion protein Currently, there is no effective treatment for retinoschisis, though viral vector-mediated gene replacement therapies offer promise We used intravitreal delivery of three different AAV vectors to target delivery of the RS1 gene to Muller glia, photoreceptors, or multiple cell types throughout the retina Muller glia radially span the entire retina, are accessible from the vitreous, and remain intact throughout progression of the disease Molecular Therapy Volume 22, Supplement 1, May 2014 Copyright © The American Society of Gene & Cell Therapy S47 ... Herewith, we demonstrate the therapeutic potential of AAV- based delivery of AONs to treat CEP290- associated LCA 125 Hybrid VEGF/PDGF Soluble Receptors for Inhibition of Ocular Neovascularization Peter... this study, we examined the feasibility of self-complementary adeno -associated virus (scAAV) vector mediated gene therapy for glaucoma and retinal artery occlusion using rat retinal ischemic... generated type mutant (triple Y-F) scAAV vector expressing BDNF (scAAV2-BDNF) and intravitreal injection was performed We used type mutant scAAV vector expressing GFP (scAAV2-GFP) as a control Two weeks