Narfström Kristina, Katz Martin L, Bragadottir Ragnheidur, Seeliger Mathias, Boulanger Ana, Redmond T Michael, Caro Lynette, Lai Chooi-May, Rakoczy P Elizabeth
Vision Science Group, Department of Veterinary Medicine and Surgery, College of Veterinary Medicine, University of Missouri-Columbia, Columbia, Missouri 65211, USA.
Invest Ophthalmol Vis Sci. 2003 Apr;44(4):1663-72. doi: 10.1167/iovs.02-0595.
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.
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.
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.
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.
评估腺相关病毒（AAV）介导的基因治疗对大量视网膜色素上皮特异性65千道尔顿蛋白（RPE65）基因敲除（-/-）犬恢复视力的疗效，并确定该治疗是否会引起全身和局部副作用。
将正常RPE65犬的互补DNA（cDNA）亚克隆到巨细胞病毒启动子控制下的重组腺相关病毒（rAAV）载体中，并制备了滴度分别为2×10¹²颗粒/毫升和2×10¹⁰转导单位/毫升的AAV.绿色荧光蛋白（GFP）对照载体。分别对4至30个月大的RPE65（-/-）犬的每只眼睛进行视网膜下注射AAV.RPE65和对照载体，对三只24至30个月大的正常对照犬的眼睛注射对照载体。进行基线及术后的全身和眼科检查、血液筛查、视力测试和视网膜电图（ERG）检查。两只RPE65（-/-）犬在治疗后3个月和6个月处死，用于视网膜的形态学检查。
RPE65（-/-）犬从出生起几乎失明，ERG不可记录或波幅很低。对28只眼睛进行了构建体注射或假手术；11只眼睛接受了AAV.RPE65构建体的视网膜下注射。术后3个月的ERG显示，在接受注射的眼睛中，暗适应b波幅平均恢复到正常的28%，明适应b波幅恢复到正常的32%。在6至9个月的随访期间，ERG波幅没有降低。未观察到全身副作用，但9只接受AAV.RPE65治疗的眼睛发生了葡萄膜炎。接受对照载体治疗的眼睛未观察到葡萄膜炎。免疫细胞化学显示，接受AAV.RPE65治疗的眼睛的视网膜色素上皮（RPE）中有RPE65表达。荧光显微镜显示，在接受AAV.GFP治疗的眼睛的RPE中以及在较小程度上在神经视网膜中有绿色荧光蛋白（GFP）表达。超微结构上，在AAV.RPE65转基因注射部位观察到RPE脂质滴积聚的逆转，而在对照载体注射部位未观察到。
在11只接受治疗的RPE65（-/-）眼睛中的10只中，基因转移导致了视力的发育，主观上表现为眼球震颤消失，客观上通过ERG记录。在结构上，RPE中的脂质滴积聚发生了逆转。75%接受转基因治疗的眼睛发生了葡萄膜炎，这一并发症可能是由于对RPE65分子的免疫致病反应所致。
