Capsid Mutated Adeno-Associated Virus Delivered to the Anterior Chamber Results in Efficient Transduction of Trabecular Meshwork in Mouse and Rat.
Capsid Mutated Adeno-Associated Virus Delivered to the Anterior Chamber Results in Efficient Transduction of Trabecular Meshwork in Mouse and Rat.
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DOI：
10.1371/journal.pone.0128759
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发表时间：
2015
期刊：
影响因子：
3.7
通讯作者：
Boye SE
中科院分区：
文献类型：
--
作者：
Bogner B;Boye SL;Min SH;Peterson JJ;Ruan Q;Zhang Z;Reitsamer HA;Hauswirth WW;Boye SE
腺相关病毒（AAV）以其将转基因递送到视网膜并介导患有遗传性视网膜疾病的动物模型和患者的改善的能力而众所周知。虽然该领域不太先进，但人们对AAV靶向眼前节细胞的能力越来越感兴趣。我们的研究的目的是充分阐明一种可靠的和可重复的方法，用于注射前房（AC）的小鼠和大鼠，并调查转导概况的AAV 2-和AAV 8-基于衣壳突变体含有自我互补（SC）基因组在眼前段的眼睛。在C57 BL/6小鼠和Sprague道利大鼠中进行AC注射。角膜在虹膜角膜角前方被刺穿。为了密封穿刺部位并防止回流，在AC中产生气泡。注射表达GFP的scAAV，并通过免疫组织化学评价转导。亲本血清型和衣壳修饰均影响表达。基于scAAV 2的载体在角膜内皮、睫状体非色素上皮（NPE）、虹膜和房角（包括小梁网）中介导有效的GFP信号，其中scAAV 2（Y 444 F）和scAAV 2（三联体）最有效。这是第一个研究，以半定量地评估前节组织的转导后注射的captain突变的AAV载体。基于scAAV 2的载体比基于scAAV 8的载体更有效地转导角膜内皮、睫状体NPE、虹膜和小梁网。表面暴露的酪氨酸残基的突变大大增强了scAAV 2在这些组织中的转导效率。然而，Y-F突变的数量与转导效率不成正比，这表明仅蛋白酶体避免可能还不够。这些结果适用于发展有针对性的，基于基因的策略，以调查前段的病理过程，并可能适用于发展基于基因的治疗青光眼和获得性或遗传性角膜异常。
Adeno associated virus (AAV) is well known for its ability to deliver transgenes to retina and to mediate improvements in animal models and patients with inherited retinal disease. Although the field is less advanced, there is growing interest in AAV’s ability to target cells of the anterior segment. The purpose of our study was to fully articulate a reliable and reproducible method for injecting the anterior chamber (AC) of mice and rats and to investigate the transduction profiles of AAV2- and AAV8-based capsid mutants containing self-complementary (sc) genomes in the anterior segment of the eye. AC injections were performed in C57BL/6 mice and Sprague Dawley rats. The cornea was punctured anterior of the iridocorneal angle. To seal the puncture site and to prevent reflux an air bubble was created in the AC. scAAVs expressing GFP were injected and transduction was evaluated by immunohistochemistry. Both parent serotype and capsid modifications affected expression. scAAV2- based vectors mediated efficient GFP-signal in the corneal endothelium, ciliary non-pigmented epithelium (NPE), iris and chamber angle including trabecular meshwork, with scAAV2(Y444F) and scAAV2(triple) being the most efficient. This is the first study to semi quantitatively evaluate transduction of anterior segment tissues following injection of capsid-mutated AAV vectors. scAAV2- based vectors transduced corneal endothelium, ciliary NPE, iris and trabecular meshwork more effectively than scAAV8-based vectors. Mutagenesis of surface-exposed tyrosine residues greatly enhanced transduction efficiency of scAAV2 in these tissues. The number of Y-F mutations was not directly proportional to transduction efficiency, however, suggesting that proteosomal avoidance alone may not be sufficient. These results are applicable to the development of targeted, gene-based strategies to investigate pathological processes of the anterior segment and may be applied toward the development of gene-based therapies for glaucoma and acquired or inherited corneal anomalies.
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