Bainbridge J W B, Mistry A, Schlichtenbrede F C, Smith A, Broderick C, De Alwis M, Georgiadis A, Taylor P M, Squires M, Sethi C, Charteris D, Thrasher A J, Sargan D, Ali R R
Department of Molecular Genetics, Institute of Ophthalmology, University College London, London, UK.
Gene Ther. 2003 Aug;10(16):1336-44. doi: 10.1038/sj.gt.3301990.
Recombinant adeno-associated virus (rAAV) vectors are attractive candidates for the treatment of inherited and acquired retinal disease. Although rAAV vectors are well characterized in rodent models, a prerequisite to their clinical application in human patients is the thorough evaluation of their efficacy and safety in intermediate animal models. In this study, we describe rAAV-2-mediated expression of GFP reporter gene in retinal cells following local vector delivery in dogs. Subretinal delivery of rAAV.CMV.GFP was performed unilaterally in eight normal dogs from 6 weeks of age. The area of retinal transduction was maximized by the optimization of surgical techniques for subretinal vector delivery by pars-plana vitrectomy and the use of fine-gauge subretinal cannulae to create multiple retinotomies. rAAV-2 vectors mediated efficient stable reporter gene expression in photoreceptors and retinal pigment epithelial cells. We found efficient transduction of cone photoreceptors in addition to rods in both the canine retina and after subretinal vector delivery in another intermediate animal model, the feline retina. GFP expression in dogs was confined to the area of the retinal bleb and was sustained in cells at this site for at least 18 months. Electroretinography demonstrated a modest reduction in global rod-mediated retinal function following subretinal delivery of rAAV.CMV.GFP. Three of the eight animals developed delayed-onset intraocular inflammation, in two cases associated with a serum antibody response to GFP protein. We conclude that rAAV-2 vectors mediate efficient sustained transgene expression in rod and cone photoreceptors following subretinal delivery in this intermediate animal model. The possibility of adverse effects including intraocular immune responses and reduced retinal function requires further investigation prior to clinical applications in patients.
重组腺相关病毒（rAAV）载体是治疗遗传性和获得性视网膜疾病的有吸引力的候选者。尽管rAAV载体在啮齿动物模型中已得到充分表征，但在人类患者中临床应用的前提是在中间动物模型中全面评估其疗效和安全性。在本研究中，我们描述了在犬局部递送载体后，rAAV-2介导的绿色荧光蛋白（GFP）报告基因在视网膜细胞中的表达。对8只6周龄的正常犬进行单侧视网膜下注射rAAV.CMV.GFP。通过优化经扁平部玻璃体切除术进行视网膜下载体递送的手术技术，并使用细径视网膜下套管进行多次视网膜切开术，使视网膜转导面积最大化。rAAV-2载体介导了光感受器和视网膜色素上皮细胞中高效稳定的报告基因表达。我们发现在犬视网膜以及在另一种中间动物模型猫视网膜中进行视网膜下载体递送后，除了视杆细胞外，视锥光感受器也能有效转导。犬体内的GFP表达局限于视网膜泡区域，并在该部位的细胞中持续至少18个月。视网膜电图显示，视网膜下注射rAAV.CMV.GFP后，整体视杆介导的视网膜功能略有下降。8只动物中有3只出现迟发性眼内炎症，其中2例与针对GFP蛋白的血清抗体反应有关。我们得出结论，在这个中间动物模型中，视网膜下递送后，rAAV-2载体介导视杆和视锥光感受器中高效持续的转基因表达。在临床应用于患者之前，包括眼内免疫反应和视网膜功能降低等不良反应的可能性需要进一步研究。
