Adeno-associated viral (AAV) vectors represent an ideal vehicle for human gene transfer. One advantage to the AAV vector system is the availability of multiple naturally occurring serotypes that provide selective tropisms for various target cells. Strategies to enhance the properties of the natural AAV isolates have been developed and can be divided into two approaches, rational design or directed evolution. The rational design approach utilizes knowledge of AAV capsids to make targeted changes to the capsid to alter transduction efficiency or specificity, while the directed evolution approach does not require a priori knowledge of capsid structure and includes random mutagenesis, capsid shuffling, or random peptide insertion. In this study, we describe the generation of novel variants for both AAV2 and AAV5 using a rational design approach and knowledge of AAV receptor binding, surface charge, and AAV capsid protein posttranslational modifications. The novel AAV2 and AAV5 variants demonstrate improved transduction properties in both the mouse retina and cornea. The translational fidelity of the novel AAV2 variant was confirmed in the context of the nonhuman primate (NHP) retina, whereas a NHP tissue explant model was established to allow the rapid assessment of translational fidelity between species for the AAV5 variants. The capsid-modified AAV2 and AAV5 variants described in this study have novel attributes that will add to the efficacy and specificity of their potential use in gene therapy for a range of human ocular diseases.

中文翻译：

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用于向视网膜和角膜有效传递基因的工程化衣壳。

腺相关病毒 (AAV) 载体是人类基因转移的理想载体。AAV 载体系统的一个优势是可以使用多种天然血清型，这些血清型可为各种靶细胞提供选择性趋向性。已经开发了增强天然 AAV 分离物特性的策略，可以分为两种方法，合理设计或定向进化。合理的设计方法利用 AAV 衣壳的知识对衣壳进行有针对性的改变以改​​变转导效率或特异性，而定向进化方法不需要先验衣壳结构的知识，包括随机诱变、衣壳改组或随机肽插入。在这项研究中，我们使用合理的设计方法和 AAV 受体结合、表面电荷和 AAV 衣壳蛋白翻译后修饰的知识描述了 AAV2 和 AAV5 新变体的生成。新型 AAV2 和 AAV5 变体在小鼠视网膜和角膜中均表现出改善的转导特性。在非人类灵长类动物 (NHP) 视网膜的背景下证实了新型 AAV2 变体的翻译保真度，而建立了 NHP 组织外植体模型以允许快速评估 AAV5 变体物种之间的翻译保真度。