Stable rAAV-mediated transduction of rod and cone photoreceptors in the canine retina
Stable rAAV-mediated transduction of rod and cone photoreceptors in the canine retina
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DOI：
10.1038/sj.gt.3301990
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发表时间：
2003-08-01
期刊：
影响因子：
5.1
通讯作者：
Ali, RR
中科院分区：
文献类型：
--
作者：
Bainbridge, JWB;Mistry, A;Ali, RR
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得到了很好的表征，但其在人类患者中临床应用的先决条件是在中间动物模型中彻底评价其功效和安全性。在这项研究中，我们描述了rAAV-2介导的GFP报告基因在视网膜细胞中的表达后，局部载体交付在狗。从6周龄开始，在8只正常犬中单侧进行rAAV、CMV、GFP的视网膜下递送。通过睫状体平坦部玻璃体切除术优化视网膜下载体递送的手术技术，并使用细规格视网膜下插管创建多个视网膜切开术，使视网膜转导区域最大化。rAAV-2载体介导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，除了在犬视网膜中的视杆细胞外，在另一种中间动物模型猫视网膜中，在视网膜下载体递送后，锥状光感受器也能有效转导。狗的GFP表达仅限于视网膜泡区域，并在该部位的细胞中持续至少18个月。视网膜电图显示在视网膜下递送rAAV. CMV. GFP后总体视杆介导的视网膜功能适度降低。8只动物中有3只发生了迟发性眼内炎症，其中2例与对GFP蛋白的血清抗体反应有关。我们的结论是，rAAV-2载体介导有效的持续转基因表达在视杆和视锥光感受器视网膜下交付后，在这个中间的动物模型。在临床应用于患者之前，需要进一步研究包括眼内免疫反应和视网膜功能降低在内的不良反应的可能性。
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.