Allocca Mariacarmela, Mussolino Claudio, Garcia-Hoyos Maria, Sanges Daniela, Iodice Carolina, Petrillo Marco, Vandenberghe Luk H, Wilson James M, Marigo Valeria, Surace Enrico M, Auricchio Alberto
Telethon Institute of Genetics and Medicine, Via P. Castellino 111, 80131 Napoli, Italy.
J Virol. 2007 Oct;81(20):11372-80. doi: 10.1128/JVI.01327-07. Epub 2007 Aug 15.
Severe inherited retinal diseases, such as retinitis pigmentosa and Leber congenital amaurosis, are caused by mutations in genes preferentially expressed in photoreceptors. While adeno-associated virus (AAV)-mediated gene transfer can correct retinal pigment epithelium (RPE) defects in animal models, approaches for the correction of photoreceptor-specific diseases are less efficient. We evaluated the ability of novel AAV serotypes (AAV2/7, AAV2/8, AAV2/9, AAV2rh.43, AAV2rh.64R1, and AAV2hu.29R) in combination with constitutive or photoreceptor-specific promoters to improve photoreceptor transduction, a limiting step in photoreceptor rescue. Based on a qualitative analysis, all AAV serotypes tested efficiently transduce the RPE as well as rod and cone photoreceptors after subretinal administration in mice. Interestingly, AAV2/9 efficiently transduces Müller cells. To compare photoreceptor transduction from different AAVs and promoters in both a qualitative and quantitative manner, we designed a strategy based on the use of a bicistronic construct expressing both enhanced green fluorescent protein and luciferase. We found that AAV2/8 and AAV2/7 mediate six- to eightfold higher levels of in vivo photoreceptor transduction than AAV2/5, considered so far the most efficient AAV serotype for photoreceptor targeting. In addition, following subretinal administration of AAV, the rhodopsin promoter allows significantly higher levels of photoreceptor expression than the other ubiquitous or photoreceptor-specific promoters tested. Finally, we show that AAV2/7, AAV2/8, and AAV2/9 outperform AAV2/5 following ex vivo transduction of retinal progenitor cells differentiated into photoreceptors. We conclude that AAV2/7 or AAV2/8 and the rhodopsin promoter provide the highest levels of photoreceptor transduction both in and ex vivo and that this may overcome the limitation to therapeutic success observed so far in models of inherited severe photoreceptor diseases.
严重的遗传性视网膜疾病，如色素性视网膜炎和莱伯先天性黑蒙，是由在光感受器中优先表达的基因突变引起的。虽然腺相关病毒（AAV）介导的基因转移可以在动物模型中纠正视网膜色素上皮（RPE）缺陷，但纠正光感受器特异性疾病的方法效率较低。我们评估了新型AAV血清型（AAV2/7、AAV2/8、AAV2/9、AAV2rh.43、AAV2rh.64R1和AAV2hu.29R）与组成型或光感受器特异性启动子相结合改善光感受器转导的能力，光感受器转导是光感受器挽救中的一个限制步骤。基于定性分析，在小鼠视网膜下给药后，所有测试的AAV血清型都能有效地转导RPE以及视杆和视锥光感受器。有趣的是，AAV2/9能有效地转导穆勒细胞。为了定性和定量地比较不同AAV和启动子的光感受器转导，我们设计了一种基于使用表达增强型绿色荧光蛋白和荧光素酶的双顺反子构建体的策略。我们发现，AAV2/8和AAV2/7介导的体内光感受器转导水平比AAV2/5高6至8倍，AAV2/5是迄今为止被认为最有效的用于光感受器靶向的AAV血清型。此外，在视网膜下注射AAV后，视紫红质启动子比其他测试的普遍存在或光感受器特异性启动子允许更高水平的光感受器表达。最后，我们表明，在体外将视网膜祖细胞分化为光感受器后，AAV2/7、AAV2/8和AAV2/9的表现优于AAV2/5。我们得出结论，AAV2/7或AAV2/8以及视紫红质启动子在体内和体外都能提供最高水平的光感受器转导，这可能克服了迄今为止在遗传性严重光感受器疾病模型中观察到的治疗成功的限制。
