The University of Iowa Institute for Vision Research, University of Iowa, Iowa City, Iowa.
Department of Ophthalmology and Visual Sciences, Carver College of Medicine, University of Iowa, Iowa City, Iowa.
Hum Gene Ther. 2019 Nov;30(11):1371-1384. doi: 10.1089/hum.2019.159. Epub 2019 Sep 26.
The identification of >100 genes causing inherited retinal degeneration and the promising results of recent gene augmentation trials have led to an increase in the number of studies investigating the preclinical efficacy of viral-mediated gene transfer. Despite success using adeno-associated viruses, many disease-causing genes, such as or , are too large to fit into these vectors. One option for large gene delivery is the family of integration-deficient helper-dependent adenoviruses (HDAds), which efficiently transduce postmitotic neurons. However, HDAds have been shown in other organ systems to elicit an immune response, and the immunogenicity of HDAds in the retina has not been characterized. In this study, HDAd serotype 5 (HDAd5) was found to successfully transduce rod and cone photoreceptors in human retinal organ cultures. The ocular inflammatory response to subretinal injection of the HDAd5 was evaluated using a rat model. Subretinal injection of HDAd5 carrying cytomegalovirus promoter-driven enhanced green fluorescent protein (HDAd5-CMVp-eGFP) elicited a robust inflammatory response by 3 days postinjection. This reaction included vitreous infiltration of ionized calcium-binding adapter molecule 1 (Iba1)-positive monocytes and increased expression of the proinflammatory protein, intercellular adhesion molecule 1 (ICAM-1). By 7 days postinjection, most Iba1-positive infiltrates migrated into the neural retina and ICAM-1 expression was significantly increased compared with buffer-injected control eyes. At 14 days postinjection, Iba1-positive cells persisted in the retinas of HDAd5-injected eyes, and there was thinning of the outer nuclear layer. Subretinal injection of an empty HDAd5 virus was used to confirm that the inflammatory response was in response to the HDAd5 vector and not due to eGFP-induced overexpression cytotoxicity. Subretinal injection of lower doses of HDAd5 dampened the inflammatory response, but also eGFP expression. Despite their larger carrying capacity, further work is needed to elucidate the inflammatory pathways involved and to identify an immunomodulation paradigm sufficient for safe and effective transfer of large genes to the retina using HDAd5.
超过 100 个基因的鉴定导致遗传性视网膜变性，最近基因增强试验的有希望的结果导致了越来越多的研究调查病毒介导的基因转移的临床前疗效。尽管使用腺相关病毒取得了成功，但许多致病基因，如 或 ，太大而无法装入这些载体。对于大基因传递的一种选择是整合缺陷型辅助依赖性腺病毒（HDAd）家族，其有效地转导有丝分裂后神经元。然而，已经在其他器官系统中表明 HDAd 引起免疫反应，并且 HDAd 在视网膜中的免疫原性尚未得到表征。在这项研究中，发现 HDAd 血清型 5（HDAd5）成功地转导了 人视网膜器官培养物中的棒状和锥状光感受器。使用大鼠模型评估了 HDAd5 亚视网膜注射的眼部炎症反应。HDAd5 携带巨细胞病毒启动子驱动的增强型绿色荧光蛋白（HDAd5-CMVp-eGFP）的亚视网膜注射在注射后 3 天引起强烈的炎症反应。这种反应包括玻璃体中离子钙结合接头分子 1（Iba1）阳性单核细胞的浸润和促炎蛋白细胞间黏附分子 1（ICAM-1）的表达增加。在注射后 7 天，大多数 Iba1 阳性浸润物迁移到神经视网膜中，并且与缓冲液注射对照眼相比，ICAM-1 表达显著增加。在注射后 14 天，Iba1 阳性细胞在 HDAd5 注射眼的视网膜中持续存在，并且外核层变薄。使用空 HDAd5 病毒进行的亚视网膜注射用于确认炎症反应是对 HDAd5 载体的反应，而不是由于 eGFP 诱导的过表达细胞毒性所致。HDAd5 的较低剂量的亚视网膜注射抑制了炎症反应，但也抑制了 eGFP 的表达。尽管它们具有更大的承载能力，但需要进一步的工作来阐明所涉及的炎症途径，并确定用于使用 HDAd5 将大基因安全有效地转移到视网膜的免疫调节范例。
