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445 Positive Responses in a Mouse Model of Leber Congenital Amaurosis after Treatment with Compacted DNA Nanoparticles Molecular Therapy Volume 18, Supplement 1, May 2010 Copyright © The American Soci[.]
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 OPHTHALMIC DISORDERS AND TECHNOLOGICAL ADVANCES injected into the subretinal space of WT mice eYFP expression was evaluated by spectral analyses and immunohistochemistry by confocal microscopy To evaluate therapeutic efcacy for retinal degeneration, nanoparticles with the Lrat gene controlled by the hVMD2 promoter were injected into the subretinal space of Lrat/mice and functional recovery was evaluated Results: Maintained and high level luciferase activity was observed by BLI from days to 200 after injection in the dosed eyes At day 277, luciferase lysate activity was over logs above background eYFP emission and protein expression were detected specically in the RPE cell layer at day 21 after injection in the dosed eyes Improvement of electroretinogram responses was observed in Lrat-/- mouse eyes treated with Lrat DNA nanoparticles compared to those dosed with saline Signicant levels of retinoids and immunohistochemical evidence of LRAT expression were detected in treated eyes as well Conclusion: Compacted-DNA nanoparticles can efciently deliver genes to the mouse retina and produce long-term and tissue-specic gene expression These data illustrate the therapeutic potential of the nanoparticles to treat various human retinal degenerative diseases 446 Adeno-Associated Virus Mediated Gene Therapy with a Rhodopsin Ribozyme for Treatment of Autosomal Dominant Retinitis Pigmentosa (Adrp) Haoyu Mao,1 Marina S Gorbatyuk,1 William W Hauswirth,1,2 Alfred S Lewin.1 Department of Molecular Genetics and Microbiology, University of Florida, Gainesville, FL; 2Department of Ophthalmology, University of Florida, Gainesville, FL Autosomal dominant retinitis pigmentosa (ADRP) is frequently caused by mutations in the RHO gene, which codes for the opsin of rod photoreceptor cells We are engaged in development of gene therapies for ADRP using adeno-associated virus (AAV), which efciently infects and transduces photoreceptor cells In the present set of studies, we designed and constructed a ribozyme (Rz1) which can efciently cut human rhodopsin (RHO) gene but not the “hardened” form of RHO The “hardened” form of RHO is a silent mutant RHO which specically resistant to degradation by Rz1 but expressing a normal RHO HEK 293 cells were co-transfected with a plasmid carrying RHO cDNA and a chemically synthesized Rz1 Reduction of RHO mRNA was detected by alkaline phosphastase activity in vitro Rz1 suppressed the RHO with 95% reduction of phosphatase activity compared with that of a control ribozyme We are testing gene therapy in a mouse model of ADRP that expresses a human transgene with a prevalent ADRP mutation: proline 23 substituted by histidine (P23H) Following AAV delivery of Rz1 to P23H transgenic mice in a background of C57BL/6 wild type mice, the retinal degeneration of injected eyes was monitored by the electroretinography (ERG), a non-invasive technique that measures the activity of photoreceptors and retinal inter neurons AAV delivery of Rz1 increased maximum a- and b-waves ERG responses, relative to eyes injected with a control virus These results encourage us to test the replacement of normal rhodopsin mRNA by the construction of an AAV vector expressing both Rz1 and ribozyme-resistant RHO (RHORz1) combination 447 POD DNA Nanoparticles Rescue an Adult Mouse Model of Retinal Degeneration Sarah P Read, Siobhan M Cashman, Rajendra Kumar-Singh Ophthalmology, Tufts University, Boston, MA Purpose: POD, or “Peptide for Ocular Delivery,” is a novel synthetic cationic cell penetrating peptide (CPP) which when modied by addition of a polyethylene glycol moiety (PEG-POD) can be used to compact plasmid DNA into 120-150 nm nanoparticles These nanoparticles can transfect the retinal pigment epithelium (RPE) Molecular Therapy Volume 18, Supplement 1, May 2010 Copyright © The American Society of Gene & Cell Therapy following delivery to the subretinal space of adult mice To test the physiological relevance of PEG-POD mediated gene delivery, PEG-POD DNA nanoparticles carrying a transgene expression cassette coding for Glial-cell line Derived Neurotrophic Factor (PEGPOD∼GDNF) were examined for their ability to rescue photoreceptors in a light induced model of retinal degeneration Methods: Four days following subretinal delivery of PEG-POD∼GDNF, PEG-POD∼Lux (nanoparticles expressing a luciferase transgene) or buffer alone, retinal degeneration was induced by exposure of mice to bright blue light for hours Expression of GDNF was conrmed using RT-PCR Apoptosis was quantied by both TUNEL and nucleosome release ELISA Anatomical and functional rescue was measured at and 14 days post-light exposure by quantication of the outer nuclear layer (ONL) thickness and by electroretinogram (ERG), respectively Results: Retinas injected with PEG-POD∼GDNF showed a signicant (∼7-fold) decrease in the number of TUNEL positive cells in the ONL relative to those injected with either PEG-POD∼Lux (p