AAV cis-regulatory sequences are correlated with ocular toxicity
AAV cis-regulatory sequences are correlated with ocular toxicity
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AAV 顺式调控序列与眼毒性相关
DOI：
10.1073/pnas.1821000116
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发表时间：
2019-03-19
影响因子：
11.1
通讯作者：
Cepko, Constance L.
中科院分区：
文献类型：
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作者：
Xiong, Wenjun;Wu, David M.;Cepko, Constance L.
腺相关病毒载体（AAV）由于其许多优点，包括与其他病毒相比其炎症特征降低，已成为基因治疗的热门。然而，即使在免疫豁免的领域，如眼睛，AAV载体能够引发宿主细胞反应。为了研究这种应答对几种眼部细胞类型的影响，我们使用视网膜下注射在小鼠中测试了多种AAV基因组结构和衣壳类型。进行形态学、炎症和生理学分析。光感受器和视网膜色素上皮（RPE）的病理影响进行了观察。Muller胶质细胞和小胶质细胞被激活，促炎细胞因子TNF-α和IL-1 β上调。顺式调控序列与毒性之间存在很强的相关性。具有三种广泛活性启动子中的任何一种或RPE特异性启动子的AAV是有毒的，而具有四种不同光感受器特异性启动子的AAV在测试的最高剂量下没有毒性。毒性与转基因、衣壳类型、制备方法或制备中的细胞污染物之间几乎没有相关性。毒性作用具有剂量依赖性，RPE比光感受器更敏感。我们的研究结果表明，眼部AAV毒性与某些AAV顺式调节序列和/或其活性相关，视网膜损伤的发生是由于RPE和/或小胶质细胞的反应。通过应用多种灵敏的毒性测定，可以设计AAV载体，使得它们可以以高剂量安全地使用，从而潜在地提供更大的治疗功效。
Adeno-associated viral vectors (AAVs) have become popular for gene therapy, given their many advantages, including their reduced inflammatory profile compared with that of other viruses. However, even in areas of immune privilege such as the eye, AAV vectors are capable of eliciting host-cell responses. To investigate the effects of such responses on several ocular cell types, we tested multiple AAV genome structures and capsid types using subretinal injections in mice. Assays of morphology, inflammation, and physiology were performed. Pathological effects on photoreceptors and the retinal pigment epithelium (RPE) were observed. Muller glia and microglia were activated, and the proinflammatory cytokines TNF-alpha and IL-1 beta were up-regulated. There was a strong correlation between cis-regulatory sequences and toxicity. AAVs with any one of three broadly active promoters, or an RPE-specific promoter, were toxic, while AAVs with four different photoreceptor-specific promoters were not toxic at the highest doses tested. There was little correlation between toxicity and transgene, capsid type, preparation method, or cellular contaminants within a preparation. The toxic effect was dose-dependent, with the RPE being more sensitive than photoreceptors. Our results suggest that ocular AAV toxicity is associated with certain AAV cis-regulatory sequences and/or their activity and that retinal damage occurs due to responses by the RPE and/or microglia. By applying multiple, sensitive assays of toxicity, AAV vectors can be designed so that they can be used safely at high dose, potentially providing greater therapeutic efficacy.