Functional and structural recovery of the retina after gene therapy in the RPE65 null mutation dog.
OBJECTIVE
To assess the efficacy of AAV-mediated gene therapy to restore vision in a large number of RPE65(-/-) dogs and to determine whether systemic and local side effects are caused by the treatment.


METHODS
Normal RPE65 dog cDNA was subcloned into an rAAV vector under control of a cytomegalovirus promoter, and an AAV.GFP control vector was also produced with the titers 2 x 10(12) particles/mL and 2 x 10(10) transducing U/mL, respectively. RPE65(-/-) dogs, aged 4 to 30 months were treated with subretinal injections of the AAV.RPE65 and control vectors, respectively, in each eye, and three 24- to 30-month-old normal control dogs with the latter. Baseline and postoperative systemic and ophthalmic examinations, blood screenings, vision testing, and electroretinography (ERG) were performed. Two RPE65(-/-) dogs were killed at 3 and 6 months after treatment for morphologic examination of the retinas.


RESULTS
RPE65(-/-) dogs were practically blind from birth with nonrecordable or low-amplitude ERGs. Construct injections or sham surgeries were performed in 28 eyes; 11 were injected subretinally with the AAV.RPE65 construct. ERGs at 3 months after surgery showed that in the latter eyes, dark-adapted b-wave amplitudes recovered to an average of 28% of normal, and light adapted b-wave amplitudes to 32% of normal. ERG amplitudes were not reduced during a 6- to 9-month follow-up. No systemic side effects were observed, but uveitis developed in nine AAV.RPE65-treated eyes. No uveitis was observed in the eyes treated with the control vector. Immunocytochemistry showed expression of RPE65 in the retinal pigment epithelium (RPE) of AAV.RPE65-treated eyes. Fluorescence microscopy showed expression of green fluorescent protein (GFP) in the RPE and, to a lesser extent, in the neural retinas of AAV.GFP-treated eyes. Ultrastructurally, a reversal of RPE lipid droplet accumulation was observed at the AAV.RPE65 transgene injection site, but not at the site of injection of the control vector.


CONCLUSIONS
In 10 of 11 treated RPE65(-/-) eyes, gene transfer resulted in development of vision, both subjectively apparent by loss of nystagmus, and objectively recorded by ERG. Structurally, there was reversal of lipid droplet accumulation in the RPE. Uveitis developed in 75% of the transgene-treated eyes, a complication possibly due to an immunopathogenic response to the RPE65 molecule.
Narfström
Kristina
K
Vision Science Group, Department of Veterinary Medicine and Surgery, College of Veterinary Medicine, University of Missouri-Columbia, Columbia, Missouri 65211, USA. narfstromk@missouri.edu
Katz
Martin L
ML
Bragadottir
Ragnheidur
R
Seeliger
Mathias
M
Boulanger
Ana
A
Redmond
T Michael
TM
Caro
Lynette
L
Lai
Chooi-May
CM
Rakoczy
P Elizabeth
PE
D000818:Animals; D003623:Dark Adaptation; D000229:Dependovirus / Q000235:genetics; D004283:Dog Diseases / Q000235:genetics / Q000503:physiopathology / Q000628:therapy*; D004285:Dogs; D004596:Electroretinography / Q000662:veterinary; D005136:Eye Proteins / Q000235:genetics*; D005260:Female; D018014:Gene Transfer Techniques / Q000662:veterinary; D015316:Genetic Therapy / Q000009:adverse effects / Q000379:methods*; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins; D007202:Indicators and Reagents / Q000378:metabolism; D050356:Lipid Metabolism; D008164:Luminescent Proteins / Q000378:metabolism; D008297:Male; D009154:Mutation; D009755:Night Blindness / Q000235:genetics / Q000503:physiopathology / Q000628:therapy / Q000662:veterinary*; D010857:Pigment Epithelium of Eye / Q000378:metabolism / Q000648:ultrastructure; D011506:Proteins / Q000235:genetics*; D012160:Retina / Q000378:metabolism / Q000473:pathology / Q000503:physiopathology*; D012162:Retinal Degeneration / Q000235:genetics / Q000503:physiopathology / Q000628:therapy / Q000662:veterinary*; D014605:Uveitis / Q000209:etiology; D014787:Vision Tests / Q000662:veterinary
D005136:Eye Proteins; D007202:Indicators and Reagents; D008164:Luminescent Proteins; D011506:Proteins; D049452:Green Fluorescent Proteins