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442 Pan Retinal Secretion of GDNF Following Intravitreal Transduction of Müller Glia Slows Photoreceptor Loss in Retinal Degeneration Molecular Therapy Volume 18, Supplement 1, May 2010 Copyright © Th[.]
OPHTHALMIC DISORDERS AND TECHNOLOGICAL ADVANCES Lipoplexes encoding LacZ were applied to either stainless steel coupons, phosphorylcholine-coated stents or cobalt chromium stents In vitro efcacy was evaluated for coronary artery smooth muscle cells from coupons whereas in vivo gene delivery was assessed in a rabbit iliac artery model The lipoplexes were characterised using electron microscopy, atomic force microscopy and for surface charge Our results show that the average diameter of lipoplex particles ranged from 30 to 75 nm with DDAB/DOPE being the smallest and POPC/ Chol being the largest Both DDAB/DOPE and POPC/Chol had only anionic charged populations whereas lipofectin had both anionic and cationic charge populations Localised liposomal gene delivery was achieved in the present study using all three lipoplexes examined In particular prolonged gene delivery out to 28 days was seen for all three formulations examined in vivo No statistical difference between delivery efcacy of the three lipoplexes was found either in vitro or in vivo despite differences in platform and biophysical proles 440 Efcient and Localized Gene Transference in Rat Heart by Electroporation Leonardo P Carvalho,1 Eduardo G Yasumura,1 Vivian Somoto,1,2 Priscila M A Denapoli,1 Flavia H Silva,1 Valderez B Valero,1 Hans F Dohmann,3 Christina M Takiya,2 Sang W Han.1 Interdisciplinary Center for Gene Therapy, Federal University of São Paulo, São Paulo, Brazil; 2Department of Histology, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil; 3Municipal Secretary of Rio de Janeiro, Rio de Janeiro, Brazil Background: The main obstacle of gene therapy to cardiovascular diseases using non-viral vectors is the inefficiency of gene transference by currently available methods Electroporation has been used successfully to transfer genes to many organs, but its use to heart is questionable because the electric shock can interfere the heart´s electromechanical functionality Methods: Male week-old Wistar rats were anesthetized and respiration of the animals was maintained with a breathing apparatus In the left parasternal area an incision was made in the fourth intercostal space to expose the heart In the left ventricle myocardium 100 µl of pSVLacZ plasmid (expresses beta-galactosidase) or empty vector containing 50, 100 or 200 µg of plasmids was injected Immediately a pair of needle electrode was inserted between the plasmid injected area and pulses of 80 V/ cm were applied One group of rats was euthanized after 48 hours for X-gal staining and other groups after weeks for histological analysis Electrocardiography (EKG) and echocardiography (ECH) was carried out before and after weeks of gene transference Results and conclusion: The mortality was less than 10% and EKG and ECH showed no signicant alterations The rats transfected with 50 µg had only few points of blue cells and those received only PBS or empty vectors had no blue cells However, the rats transfected with 100 or 200 µg had many blue spots in hearts, especially at the local of electroporation Histological analysis showed no visible tissue alterations, except for few localized brosis at the places where the needle electrodes were inserted By this study we conclude that the electroporation of rat heart to gene transference is safe and efcient to transfection Molecular Therapy Volume 18, Supplement 1, May 2010 Copyright © The American Society of Gene & Cell Therapy Ophthalmic Disorders and Technological Advances 441 Gene Delivery of Erythropoietin Protects Retinal Ganglion Cells in DBA/2J Glaucomatous Mice Timothy A Sullivan,1 Eldon E Geisert,1 Tonia S Rex.1 Opthalmology, University of Tennessee, Memphis, TN Background: Classically one thinks of erythropoietin (EPO) as a regulator of red blood cell production Recent evidence points to EPO as a putative neuroprotective agent EPO and its receptor are present in selected CNS neurons including retinal ganglion cells (RGCs) A modied form of EPO containing a single arginine to glutamate mutation (R103E) is neuroprotective while not triggering erythropoiesis The goal of this study was to determine if systemic delivery of EPO-R103E would protect the RGCs from cell death in a mouse model of pigmentary dispersion glaucoma (DBA/2J mouse) Methods: One month old DBA/2J mice received intramuscular injections of a recombinant adeno-associated virus (rAAV) carrying either eGFP, Epo or EpoR103E under the control of the cytomegalovirus promoter Intraocular pressure was measured at six months to detect elevated pressure At ten months, isolated retinas were stained with NueN, at-mounted, and examined at 40X by confocal microscopy Hematocrit levels were determined by capillary centrifugation of blood and serum EPO levels were determined by ELISA Results: Retinas from DBA/2J mice that received either rAAV.Epo or rAAV.EpoR103E had an average of 30% more cells present at the RGC layer then control mice (rAAV.eGFP) Hematocrit levels were not substantially increased in the rAAV.Epo R103E treated mice as compared to rAAV.Epo treated mice Conclusion: A single systemic injection of rAAV.Epo protects RGCs from glaucomatous cell death, but also increases the hematocrit to harmful levels Treatment with rAAV.EpoR103E provides comparable levels of RGC protection but does not signicantly increase the hematocrit Thus, a single dose of rAAV.EpoR103E can provide long-term protection without the need for intraocular injections and without elevating the hematocrit 442 Pan-Retinal Secretion of GDNF Following Intravitreal Transduction of Müller Glia Slows Photoreceptor Loss in Retinal Degeneration Deniz Dalkara,1 Kathleen D Kolstad,2 Meike Visel,3 Ryan R Klimczak,2 Karen Guerin,4 David V Schaffer,1 John G Flannery.2 HWNI and CChem, UC Berkeley, Berkeley, CA; 2Molecular and Cellular Biology, UC Berkeley, Berkeley; 3HWNI, UC HWNI, HWNI; 4Vision Science, UC Vision Science, Vision Science Gene therapy via secretion of neurotrophic factors (NFs) has signicant potential for treating many retinal degenerations without requiring knowledge of the specic disease mechanism In retinal degenerations, Müller glia (MG) survive late in the disease, and their close physical contact with all retinal cell types makes them strong candidates for secreting NF or anti-angiogenic factors In addition, MG extend radially through the retina, allowing viral transduction from their vitreal endfeet, which avoids the detachment-related trauma of subretinal injections Furthermore, intravitreal injection yields broad pan-retinal expression not available with a focal subretinal ‘bleb’ We engineered a MG-specic AAV vector capable of high-efciency MG transduction from the vitreous and explored its therapeutic potential This AAV vector promotes secretion of the neurotrophin GDNF from MG in a transgenic rat model of retinitis pigmentosa The GDNF protein concentration in retinal homogenates and vitreal uid of injected animals was ∼2500 pg/ml, (10-fold greater than previous studies of GDNF-induced rescue) Furthermore, signicant anatomic and functional improvement was observed S171 OPHTHALMIC DISORDERS AND TECHNOLOGICAL ADVANCES by histopathology and ERG in eyes treated with ShHY10- GDNF Moreover, this rescue persisted for a signicantly longer duration (>5 months post-injection) than previous studies The GDNF protein concentration in retinal homogenates and vitreal uid of injected animals was ∼2500 pg/ml, (10-fold greater than previous studies of GDNF-induced rescue) Furthermore, signicant anatomic and functional improvement was observed by histopathology and ERG in eyes treated with ShHY10- GDNF Moreover, this rescue persisted for a signicantly longer duration (>5 months post-injection) than previous studies Neuroprotection of photoreceptors by GDNF overexpression from MG is a highly promising strategy to slow retinal degeneration The use of MG-specic AAV from the vitreous is advantageous in terms of safety, efcacy, and longevity Therefore, the less invasive intravitreal approach has potential for the treatment of retinal degenerations of known and unknown etiology 443 AAV-Mediated Gene Supply of NonErythropoietic Erythropoietin Derivatives Improves Photoreceptor Morphology and Function in Models of Retinal Degenerative Diseases Pasqualina Colella,1,2 Carolina Iodice,1 Umberto Di Vicino,1 Alberto Auricchio.1,3 Telethon Institute of Genetics and Medicine (TIGEM), Naples, Italy; 2The Open University, Milton Keynes, United Kingdom; Medical Genetics, Dept of Pediatrics, Federico II University, Naples, Italy Gene supply of neurotrophic/antiapoptotic factors provides a mutation-independent therapy for inherited retinal degenerations (IRDs) that is desirable given their high genetic heterogeneity In addition photoreceptor cells preservation achieved by gene supply may allow subsequent gene-specic therapy The cytokine Erythropoietin (EPO) has shown promising neurotrophic and antiapoptotic effects in several models of neuronal degeneration including IRDs as we have previously reported that systemic adeno-associated viral (AAV) vector-mediated EPO delivery protects animal models from photoreceptor degeneration Translation of these findings into therapeutic application looks promising, however the EPO erythrodifferentiating function causes several undesired side effects Recently, EPO derivatives that not increase hematocrits but retain tissue-protective and anti-apoptotic functions have been identied We are currently investigating the potential neuroprotective effects of the EPO mutant S100E (EpoS100E) and of short EPO-derived peptides following AAV2/1 intramuscular and intraocular delivery in animal models of light-induced and inherited retinal degenerations High EpoS100E levels were measured in the sera and ocular uids of albino rats administered systemically and subretinally, respectively Rats treated with EpoS100E but not with the Epo peptides had a minor increase in hematocrits After light-damage, animals administered either systemically or intraocularly with AAV1-CMV-EpoS100E orEpo peptides showed increased photoreceptor survival and preserved retinal function although to a lesser extent compared to wild type EPO Systemic delivery of Epo or EpoS100E or intraocular delivery of Epo peptides were most effective We are currently testing EpoS100E and EPO-derived neurotrophic peptides in the Aipl1-/- and Phrp2rd2/rd2 animal models of IRDs In conclusion, our data support the neuroprotective effect of non erythropoietic Epo derivatives in retinal degenerations while conrming an important role of EPO erythropoietic activity in photoreceptor protection S172 444 Early Indicators of Biological Efcacy in a Phase 1/2 Gene Therapy Trial for Leber Congenital Amaurosis (LCA) Shalesh Kaushal,1 Margaret Humphries,1 Elena Filippova,1 George Asdourian,1 Radouil Tzekov,1 Michael Stalvey,2 Terrence Flotte.2 Ophthalmology, University of Massachusetts School of Medicine, Worcester, MA; 2Pediatrics, University of Massachusetts School of Medicine, Worcester, MA A number of recent reports have demonstrated long-term improvements in photoreceptor function in patients with Leber Congenital Amaurosis (LCA) due to mutations of RPE65 after subretinal injection of recombinant adeno-associated virus serotype (rAAV2-hRPE65) vectors Our group initiated a phase 1/2 study of one of these vectors (rAAV2-CB-hRPE65) in July 2009, anticipating dosing a total of 12 participants (4 groups of 3) Group will consist of adults (18 yrs and older) receiving 1.8x1011vg in 450 µls Group will include children to 17 years old at that same dose Groups and will consist of adults and children, respectively, receiving 6.0x1011vg these doses and volume represent a step forward to a clincially relevant dosing scheme Safety outcomes will include monitoring for both ocular and non-ocular toxicities and efcacy measures including visual elds, visual acuity and electroretinography In the rst treated patient, safety assessment from the rst months after injection have indicated only mild transient adverse events, consisting of the sensation of bright ashes of light and some conjunctival congestion Efcacy measures indicate an increase in visual elds and subjective improvements in visual function Further enrollment and follow-on results are anticipated 445 Positive Responses in a Mouse Model of Leber Congenital Amaurosis after Treatment with Compacted DNA Nanoparticles Kiichiro Okano,3 Tadao Maeda,1,3 Linas Padegimas,2 Tomasz H Kowalczyk,2 Sharon M Oette,2 Christopher R Gedeon,2 Ozge Sesenoglu-Laird,2 Susannah L Hyatt,2 Karla A Dines,2 Elena Tyr,2 Nikia L Beal,2 Robert C Moen,2 Krzysztof Palczewski,3 Mark J Cooper.2 Ophthalmology, Case Western Reserve University, Cleveland, OH; Copernicus Therapeutics, Inc., Cleveland, OH; 3Pharmacology, Case Western Reserve University, Cleveland, OH Purpose: Compacted-DNA nanoparticles are a highly-efcient and non-toxic non-viral gene delivery system Human vision is initiated by photon absorption in visual pigments of retinal photoreceptors in the eye Continuous regeneration of visual pigment 11-cis-retinal depends on an enzymatic pathway in photoreceptor and retinal pigmented epithelial (RPE) cells known as the visual cycle, which is essential to maintain human vision Dysfunction of the visual cycle due to DNA mutation causes various inherited-retinal degenerative diseases, such as Leber congenital amaurosis (LCA) and retinitis pigmentosa (RP) The purpose of this study is to evaluate the efciency and specicity of gene delivery by the nanoparticles in the wild-type (WT) mouse retina and the restoration of visual function in an LCA mouse model, the lecithin:retinol acyl transferase (LRAT) gene-decient mouse (Lrat-/-), which is lacking 11-cis-retinal and other retinoid intermediates in the eye Methods: To evaluate duration of functional gene expression by nanoparticle-mediated gene delivery, nanoparticles containing the luciferase gene controlled by the polyubiquitin C promoter were injected into the subretinal space of WT mice The enzymatic activity of luciferase was visualized and quantied by Bioluminescent Imaging (BLI) Luciferase activity in mouse eye lysates was evaluated by a biochemical assay To examine gene delivery to specic target cells in the mouse retina, nanoparticles containing the enhanced yellow uorescent protein (eYFP) gene, transcriptionally controlled by the RPE cell-specic human VMD2 promoter (hVMD2), were Molecular Therapy Volume 18, Supplement 1, May 2010 Copyright © The American Society of Gene & Cell Therapy ... vision is initiated by photon absorption in visual pigments of retinal photoreceptors in the eye Continuous regeneration of visual pigment 11-cis -retinal depends on an enzymatic pathway in photoreceptor. .. mutant S100E (EpoS100E) and of short EPO-derived peptides following AAV2/1 intramuscular and intraocular delivery in animal models of light-induced and inherited retinal degenerations High EpoS100E... highly promising strategy to slow retinal degeneration The use of MG-specic AAV from the vitreous is advantageous in terms of safety, efcacy, and longevity Therefore, the less invasive intravitreal