AAV Induced Toxicity in the Eye

项目总结/摘要 AAV载体已成为将基因递送至多种组织的主要载体，证明其既安全又安全。 并且在几个临床试验中有效。然而，它们的安全极限尚未得到充分探索 效能由于AAV介导的基因治疗的治疗益处几乎总是随着基因治疗的进展而增加， 当转导更多数量的细胞时，高病毒剂量的安全递送将可能提供更大的 对患者有益。然而，在非人灵长类动物（NHP）和其他动物模型中的高AAV剂量具有以下作用： 与毒性有关。如果这些病毒引起的问题的机制可以阐明，它可能是 我们可以避免它们，以便更大的病毒剂量可以安全地输送。小鼠模型可以用于研究 然后可以在更大的动物身上进行研究。我们已经发现AAV载体可以伤害 眼细胞，特别是小鼠、猪和小鼠中的视锥光感受器细胞和视网膜色素上皮（RPE）， 狗这种毒性可导致视网膜神经元和RPE的损失。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， 剂量和基因组序列我们建议确定导致和/或保护， 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，以识别细胞 启动毒性的细胞类型，以及可能放大病毒检测结果的细胞类型。到 为此，我们将探讨几种眼细胞类型在感染毒性角膜炎后随时间的RNA变化。 和无毒的AAV制剂。我们还将跟踪这些基因触发的机制 表达变化，并寻求阻止它们的方法。这些发现将扩展到大脑，使用 将有毒和无毒的原料注射到大脑皮层。我们将分析大脑RNA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， 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.