- Email: [email protected]
885. AAV-Mediated Gene Transfer of Constitutively and Pharmacologically-Activated Glial Cell-Derived Neurotrophic Factor Receptor To Inhibit Retinal Degeneration
NEUROLOGIC: APPLICATIONS TO BRAIN AND EYE; VECTOR DEVELOPMENT; IMMUNE RESPONSES protection, which increased the viability ratio almost two-fold, the greatest protection with an anti-oxidant gene was observed with CuZnSOD expression. A similar trend was also observed in the reduction of apoptotic cells with CuZnSOD demonstrating increased numbers of viable cells. No protection was observed with HO-1 treatment but interestingly HSP-27 treatment demonstrated a level of protection significantly greater than any anti-oxidant treatment. These in-vitro results demonstrate a potential therapeutic benefit from expression of either CuZnSOD or HSP27 in AMD.
885. AAV-Mediated Gene Transfer of Constitutively and Pharmacologically-Activated Glial Cell-Derived Neurotrophic Factor Receptor To Inhibit Retinal Degeneration Mariacarmela Allocca,1,2 Umberto Di Vicino,1 Luciano Domenici,3,4 Victor M. Rivera,5 Alberto Auricchio.1,6 1 Gene Therapy, TIGEM (Telethon Institute of Genetics and Medicine), Naples, Italy; 2S.E.M.M., European School of Molecular Medicine, Naples, Italy; 3Elettrofisiologia, Istituto di Neuroscienze del C.N.R., Pisa, Italy; 4Cognitive Neuroscience Sector, International School for Advanced Studies, Trieste, Italy; 5 Pharmacology, ARIAD Pharmaceuticals, Inc, Cambridge, MA; 6 Dept of Pediatrics, “Federico II” University, Naples, Italy. Retinitis pigmentosa (RP) is a group of inherited retinal degenerations for which no treatment is available. Despite their high genetic heterogeneity, common mechanisms like apoptosis are responsible for photoreceptor cell death. Various molecules with neurotrophic activity are being evaluated for treatment of RP in animal models. Delivery of Glial Cell-Derived Neurotrophic Factor (GDNF), either as a recombinant protein or by retinal gene transfer results in photoreceptor protection in genetic models of RP. The GDNF receptor is a complex including both GFR-α and Ret, which is endowed with tyrosine kinase activity and is expressed on photoreceptor outer segments as well as in other retinal cell types. It is therefore unclear whether GDNF neuroprotective effect is exerted directly on photoreceptors. To assess GDNF neuroprotective mechanism and to test a potential therapeutic strategy to regulate its neurotrophic activity, we have developed adeno-associated viral vectors (AAV) expressing either a constitutively activated (RetMen2A) or a pharmacologically-activated chimeric Ret receptor (Fv2Ret). Fv2Ret homodimerization, trans-phosphorylation and activation are induced by administration of the small dimerizer drug AP20187. RetMen2A and Fv2Ret expression in the retina transduced with AAV 2/5 vectors is restricted to photoreceptors by the human rhodopsin promoter. Following subretinal administration of AAV2/ 5 vectors in Balb/c mice, we show recombinant Ret expression and phosphorylation which is constitutive and robust in the case of RetMen2A and dependent upon AP20187 administration, albeit at low levels, in the case of Fv2Ret. No functional or morphological protection from light-damage was observed following AAV mediated gene transfer of either Ret molecule suggesting that photoreceptors might not be the direct target of GDNF neuroprotective effect in light-induced retinal degeneration.
886. AAV-Mediated Somatic Gene Transfer as an Approach To Delineate Pathogenic Mechanisms in an Autosomal Dominant Blindness Disorder Resembling Age-Related Macular Degeneration (AMD) Nicholas W. Keiser,1 Arvydas Maminishkis,2 Jeannette L. Bennicelli,1 Sheldon S. Miller,2 Jean Bennett.1 1 Department of Ophthalmology, F.M. Kirby Center for Molecular Ophthalmology, Scheie Eye Institute, University of Pennsylvania, Philadelphia, PA; 2National Eye Institute, National Institutes of Health, Bethesda, MD. Malattia Leventinese (ML) is an autosomal dominant inherited macular degeneration with a middle-age onset. Phenotypically, the disease closely resembles Age-related Macular Degeneration (AMD), a condition affecting 20% of the population over age 65 and the leading cause of blindness in the elderly in the Western world. Patients with ML suffer from decreased visual acuity and have a characteristic radial pattern of deposits, called drusen, which form beneath the retinal pigment epithelium (RPE). A single missense mutation (R345W) in the Epidermal Growth Factor Containing Fibrillin-like Extracellular Matrix Protein 1 (EFEMP1) gene is responsible for the disease, and leads to the misfolding and inefficient secretion of the protein. In normal retinas, the protein localizes to the inner photoreceptor matrix (IPM), apical to the RPE, while in individuals with ML, it resides between the RPE and drusen. We therefore surmised that wild-type EFEMP1 is apically secreted by the RPE, and that the R345W mutation misdirects this secretion basolaterally. To test this hypothesis, we utilized a cell culture system consisting of polarized monolayers of human fetal RPE (hfRPE) cells grown on Transwell membranes. AAV2/1 vectors containing FLAG-tagged wild-type or mutant EFEMP1 were used to transduce the hfRPE cells with high efficiency. Transduction did not affect hfRPE cell polarization or tight junction formation, as shown by immunofluorescence staining for Na+/K+ ATPase, CD147, and ZO-1. Immunoprecipitation of EFEMP1-FLAG from the basolateral and apical medium revealed that, contrary to our hypothesis, EFEMP1 is secreted basolaterally, and that this polarized secretion pattern is not altered by the R345W mutation. In addition, we found that this pattern of secretion was distinct from dog epithelial (MDCK) cells, which secreted the protein apically. Additional immunoprecipitation and immunofluorescence experiments indicated that the R345W mutation resulted in retention of EFEMP1 within polarized RPE cells. We conclude that EFEMP1 is directionally secreted from epithelial cells, and that its basolateral secretion from RPE cells may be indicative of some yet unknown function in the extracellular matrix underlying those cells, within the local environment of drusen formation in ML patients. However, the absence of an alteration in polarized secretion suggests that the intracellular retention of mutant EFEMP1 may be a major contributing factor to the progression of ML.
887. G Protein-Coupled Receptor Kinase Interacting Protein 1 Slows down the Apoptosis Process of Retinal Ganglion Cells in a Rat Model Ann-Shung Lieu,1 Yi Wang,1 Donna Webb,2 Gerold R. Hankins,1 Gergory A. Helm,1,3 Jin Zhong Li.1 1 Neurological Surgery, University of Virginia, Charlottsville, VA; 2 Cell Biology, University of Virginia, Charlottsville, VA; 3 Biomedical Engineering, University of Virginia, Charlottsville, VA. Central nervous system (CNS) trauma, stoke, or neuron degeneration diseases can lead to permanent paralysis or other loss of function. However, it is becoming clear that the CNS is not
Molecular Therapy Volume 13, Supplement 1, May 2006 Copyright The American Society of Gene Therapy
S341
885. AAV-Mediated Gene Transfer of Constitutively and Pharmacologically-Activated Glial Cell-Derived Neurotrophic Factor Receptor To Inhibit Retinal Degeneration Mariacarmela Allocca,1,2 Umberto Di Vicino,1 Luciano Domenici,3,4 Victor M. Rivera,5 Alberto Auricchio.1,6 1 Gene Therapy, TIGEM (Telethon Institute of Genetics and Medicine), Naples, Italy; 2S.E.M.M., European School of Molecular Medicine, Naples, Italy; 3Elettrofisiologia, Istituto di Neuroscienze del C.N.R., Pisa, Italy; 4Cognitive Neuroscience Sector, International School for Advanced Studies, Trieste, Italy; 5 Pharmacology, ARIAD Pharmaceuticals, Inc, Cambridge, MA; 6 Dept of Pediatrics, “Federico II” University, Naples, Italy. Retinitis pigmentosa (RP) is a group of inherited retinal degenerations for which no treatment is available. Despite their high genetic heterogeneity, common mechanisms like apoptosis are responsible for photoreceptor cell death. Various molecules with neurotrophic activity are being evaluated for treatment of RP in animal models. Delivery of Glial Cell-Derived Neurotrophic Factor (GDNF), either as a recombinant protein or by retinal gene transfer results in photoreceptor protection in genetic models of RP. The GDNF receptor is a complex including both GFR-α and Ret, which is endowed with tyrosine kinase activity and is expressed on photoreceptor outer segments as well as in other retinal cell types. It is therefore unclear whether GDNF neuroprotective effect is exerted directly on photoreceptors. To assess GDNF neuroprotective mechanism and to test a potential therapeutic strategy to regulate its neurotrophic activity, we have developed adeno-associated viral vectors (AAV) expressing either a constitutively activated (RetMen2A) or a pharmacologically-activated chimeric Ret receptor (Fv2Ret). Fv2Ret homodimerization, trans-phosphorylation and activation are induced by administration of the small dimerizer drug AP20187. RetMen2A and Fv2Ret expression in the retina transduced with AAV 2/5 vectors is restricted to photoreceptors by the human rhodopsin promoter. Following subretinal administration of AAV2/ 5 vectors in Balb/c mice, we show recombinant Ret expression and phosphorylation which is constitutive and robust in the case of RetMen2A and dependent upon AP20187 administration, albeit at low levels, in the case of Fv2Ret. No functional or morphological protection from light-damage was observed following AAV mediated gene transfer of either Ret molecule suggesting that photoreceptors might not be the direct target of GDNF neuroprotective effect in light-induced retinal degeneration.
886. AAV-Mediated Somatic Gene Transfer as an Approach To Delineate Pathogenic Mechanisms in an Autosomal Dominant Blindness Disorder Resembling Age-Related Macular Degeneration (AMD) Nicholas W. Keiser,1 Arvydas Maminishkis,2 Jeannette L. Bennicelli,1 Sheldon S. Miller,2 Jean Bennett.1 1 Department of Ophthalmology, F.M. Kirby Center for Molecular Ophthalmology, Scheie Eye Institute, University of Pennsylvania, Philadelphia, PA; 2National Eye Institute, National Institutes of Health, Bethesda, MD. Malattia Leventinese (ML) is an autosomal dominant inherited macular degeneration with a middle-age onset. Phenotypically, the disease closely resembles Age-related Macular Degeneration (AMD), a condition affecting 20% of the population over age 65 and the leading cause of blindness in the elderly in the Western world. Patients with ML suffer from decreased visual acuity and have a characteristic radial pattern of deposits, called drusen, which form beneath the retinal pigment epithelium (RPE). A single missense mutation (R345W) in the Epidermal Growth Factor Containing Fibrillin-like Extracellular Matrix Protein 1 (EFEMP1) gene is responsible for the disease, and leads to the misfolding and inefficient secretion of the protein. In normal retinas, the protein localizes to the inner photoreceptor matrix (IPM), apical to the RPE, while in individuals with ML, it resides between the RPE and drusen. We therefore surmised that wild-type EFEMP1 is apically secreted by the RPE, and that the R345W mutation misdirects this secretion basolaterally. To test this hypothesis, we utilized a cell culture system consisting of polarized monolayers of human fetal RPE (hfRPE) cells grown on Transwell membranes. AAV2/1 vectors containing FLAG-tagged wild-type or mutant EFEMP1 were used to transduce the hfRPE cells with high efficiency. Transduction did not affect hfRPE cell polarization or tight junction formation, as shown by immunofluorescence staining for Na+/K+ ATPase, CD147, and ZO-1. Immunoprecipitation of EFEMP1-FLAG from the basolateral and apical medium revealed that, contrary to our hypothesis, EFEMP1 is secreted basolaterally, and that this polarized secretion pattern is not altered by the R345W mutation. In addition, we found that this pattern of secretion was distinct from dog epithelial (MDCK) cells, which secreted the protein apically. Additional immunoprecipitation and immunofluorescence experiments indicated that the R345W mutation resulted in retention of EFEMP1 within polarized RPE cells. We conclude that EFEMP1 is directionally secreted from epithelial cells, and that its basolateral secretion from RPE cells may be indicative of some yet unknown function in the extracellular matrix underlying those cells, within the local environment of drusen formation in ML patients. However, the absence of an alteration in polarized secretion suggests that the intracellular retention of mutant EFEMP1 may be a major contributing factor to the progression of ML.
887. G Protein-Coupled Receptor Kinase Interacting Protein 1 Slows down the Apoptosis Process of Retinal Ganglion Cells in a Rat Model Ann-Shung Lieu,1 Yi Wang,1 Donna Webb,2 Gerold R. Hankins,1 Gergory A. Helm,1,3 Jin Zhong Li.1 1 Neurological Surgery, University of Virginia, Charlottsville, VA; 2 Cell Biology, University of Virginia, Charlottsville, VA; 3 Biomedical Engineering, University of Virginia, Charlottsville, VA. Central nervous system (CNS) trauma, stoke, or neuron degeneration diseases can lead to permanent paralysis or other loss of function. However, it is becoming clear that the CNS is not
Molecular Therapy Volume 13, Supplement 1, May 2006 Copyright The American Society of Gene Therapy
S341