AAV Induced Toxicity in the Eye

项目摘要/摘要 AAV载体已经成为向多个组织传递基因的主要载体，事实证明这两种载体都是安全的 在几个临床试验中都是有效的。然而，它们的安全限度还没有得到充分的探索 和效率。由于AAV介导的基因治疗的治疗益处几乎总是随着 转导更多的细胞，安全传递高剂量的病毒可能会提供更大的 造福于患者。然而，在非人类灵长类动物(NHP)和其他动物模型中，高剂量的AAV会 都与毒性有关。如果这些由病毒引起的问题的机制能够被阐明，它可能是 尽可能地避免它们，以便能够安全地传递更大剂量的病毒。小鼠模型可用于研究 然后可以在更大的动物身上进行研究的机制。我们已经发现AAV媒介可以危害 眼细胞，特别是小鼠、猪和小鼠的视锥感光细胞和视网膜色素上皮(RPE) 狗。这种毒性会导致视网膜神经元和RPE的丧失。毒性与衣壳类型无关， 原料的“清洁度”，表达的基因，或制备方法。相反，毒性跟踪 剂量和基因组序列。我们建议识别引起和/或保护不受影响的序列 这种毒性。我们还计划跟踪细胞对这些有毒基因组序列的反应，以确定细胞 启动毒性的细胞类型(S)，以及可能放大病毒检测结果的细胞类型。至 为此，我们将探索几种眼细胞类型在感染有毒物质后随时间的RNA变化 和无毒的AAV制剂。我们还将遵循这些基因触发的机制(S) 表情变化，并想方设法阻止它们。这些发现将扩展到大脑，使用 将有毒和无毒的股票注入大脑皮层。我们将检测大脑核糖核酸和细胞的变化 健康，使用我们在视网膜中发现的变化作为探针。我们还将测试这一机制是否 减轻视网膜和大脑的毒性。

PROJECT SUMMARY / ABSTRACT AAV vectors have emerged as the leading vector for gene delivery to multiple tissues, proving to be both safe and efficacious in several clinical trials. However, they have not been fully explored for the limits of their safety and efficiency. As the therapeutic benefits of AAV mediated gene therapy will almost always increase with transduction of a greater number of cells, the safe delivery of high viral doses will likely provide a greater benefit to patients. However, high AAV doses in non-human primates (NHPs) and in other animal models have been associated with toxicity. If the mechanisms of these virus-induced problems can be elucidated, it may be possible to avoid them, so that a greater virus dose can be safely delivered. A mouse model allows for studies of mechanism that can then be investigated in larger animals. We have found that AAV vectors can harm ocular cells, in particular, cone photoreceptor cells and the retinal pigment epithelium (RPE) in mice, pigs, and dogs. This toxicity can lead to loss of retinal neurons and the RPE. Toxicity does not correlate with capsid type, the “cleanliness” of the stock, the gene that is expressed, or the preparation method. Rather, toxicity tracks with dose and genome sequence. We propose to identify the sequences that cause, and/or protect, against this toxicity. We also plan to track the cellular response to these toxic genomic sequences, to identify the cell type(s) in which toxicity is initiated, as well as the cell types which may amplify the results of viral detection. To this end, we will explore the RNA changes in several ocular cell types over time following infection with toxic and non-toxic AAV preparations. We will also follow the mechanism(s) that are triggered by these gene expression changes, and seek ways to block them. These findings will be extended to the brain, using injections of toxic and non-toxic stocks into the cerebral cortex. We will assay changes in brain RNA and cell health, using as probes the changes that we find in the retina. We will also test whether mechanisms that alleviate toxicity in the retina and in the brain.