Reversal of blindness in animal models of Leber congenital amaurosis using optimized AAV2-mediated gene transfer
Reversal of blindness in animal models of Leber congenital amaurosis using optimized AAV2-mediated gene transfer
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DOI：
10.1038/sj.mt.6300389
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发表时间：
2008-03-01
影响因子：
12.4
通讯作者：
Bennett, Jean
中科院分区：
文献类型：
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作者：
Bennicelli, Jeannette;Wright, John Fraser;Bennett, Jean
我们评估了一种优化的腺相关病毒（AAV; AAV2）的安全性和有效性。RPE65型Leber先天性黑朦（LCA）的动物模型。通过在hRPE65的翻译起始位点添加修饰的Kozak序列来优化蛋白表达。对AAV生产和递送的改进包括使用长填充序列来防止AAV反末端重复序列的反向包装，以及与表面活性剂共注射。后者允许持续和可预测地递送给定剂量的载体。我们观察到Rpe65突变小鼠视网膜电图（ERGs）和视力的改善。这在以前使用AAV2载体时未见报道。视网膜下注射8.25 x 10（10）个载体基因组对患病犬的局部和全身耐受良好，注射后2周内，治疗犬的视觉行为和瞳孔反应得到改善，眼球震颤减少。ERG反应证实了视觉缺陷的逆转。免疫组织化学证实了视网膜色素上皮细胞的转导，组织病理学分析表明对视网膜的毒性很小。数据表明，AAV2。在动物模型中，RPE65高效、快速地将RPE65转基因传递到体内合适的靶细胞。该载体在治疗RPE65突变引起的LCA方面具有很大的前景。
We evaluated the safety and efficacy of an optimized adeno-associated virus (AAV; AAV2.RPE65) in animal models of the RPE65 form of Leber congenital amaurosis (LCA). Protein expression was optimized by addition of a modified Kozak sequence at the translational start site of hRPE65. Modifications in AAV production and delivery included use of a long stuffer sequence to prevent reverse packaging from the AAV inverted-terminal repeats, and co-injection with a surfactant. The latter allows consistent and predictable delivery of a given dose of vector. We observed improved electroretinograms (ERGs) and visual acuity in Rpe65 mutant mice. This has not been reported previously using AAV2 vectors. Subretinal delivery of 8.25 x 10(10) vector genomes in affected dogs was well tolerated both locally and systemically, and treated animals showed improved visual behavior and pupillary responses, and reduced nystagmus within 2 weeks of injection. ERG responses confirmed the reversal of visual deficit. Immunohistochemistry confirmed transduction of retinal pigment epithelium cells and there was minimal toxicity to the retina as judged by histopathologic analysis. The data demonstrate that AAV2. RPE65 delivers the RPE65 transgene efficiently and quickly to the appropriate target cells in vivo in animal models. This vector holds great promise for treatment of LCA due to RPE65 mutations.