Matet Alexandre, Kostic Corinne, Bemelmans Alexis-Pierre, Moulin Alexandre, Rosolen Serge G, Martin Samia, Mavilio Fulvio, Amirjanians Vazrik, Stieger Knut, Lorenz Birgit, Behar-Cohen Francine, Arsenijevic Yvan
Unit of Retinal Degeneration and Regeneration, Department of Ophthalmology, University of Lausanne, Jules-Gonin Eye Hospital, Fondation Asile des Aveugles, Lausanne, Switzerland.
Unit of Retinal Degeneration and Regeneration, Department of Ophthalmology, University of Lausanne, Jules-Gonin Eye Hospital, Fondation Asile des Aveugles, Lausanne, Switzerland.
Transl Res. 2017 Oct;188:40-57.e4. doi: 10.1016/j.trsl.2017.06.012. Epub 2017 Jul 8.
Several approaches have been developed for gene therapy in RPE65-related Leber congenital amaurosis. To date, strategies that have reached the clinical stages rely on adeno-associated viral vectors and two of them documented limited long-term effect. We have developed a lentiviral-based strategy of RPE65 gene transfer that efficiently restored protein expression and cone function in RPE65-deficient mice. In this study, we evaluated the ocular and systemic tolerances of this lentiviral-based therapy (LV-RPE65) on healthy nonhuman primates (NHPs), without adjuvant systemic anti-inflammatory prophylaxis. For the first time, we describe the early kinetics of retinal detachment at 2, 4, and 7 days after subretinal injection using multimodal imaging in 5 NHPs. We revealed prolonged reattachment times in LV-RPE65-injected eyes compared to vehicle-injected eyes. Low- (n = 2) and high-dose (n = 2) LV-RPE65-injected eyes presented a reduction of the outer nuclear and photoreceptor outer segment layer thickness in the macula, that was more pronounced than in vehicle-injected eyes (n = 4). All LV-RPE65-injected eyes showed an initial perivascular reaction that resolved spontaneously within 14 days. Despite foveal structural changes, full-field electroretinography indicated that the overall retinal function was preserved over time and immunohistochemistry identified no difference in glial, microglial, or leucocyte ocular activation between low-dose, high-dose, and vehicle-injected eyes. Moreover, LV-RPE65-injected animals did not show signs of vector shedding or extraocular targeting, confirming the safe ocular restriction of the vector. Our results evidence a limited ocular tolerance to LV-RPE65 after subretinal injection without adjuvant anti-inflammatory prophylaxis, with complications linked to this route of administration necessitating to block this transient inflammatory event.
针对与RPE65相关的莱伯先天性黑蒙，已经开发了几种基因治疗方法。迄今为止，已进入临床阶段的策略依赖于腺相关病毒载体，其中两种显示出有限的长期效果。我们开发了一种基于慢病毒的RPE65基因转移策略，该策略在RPE65缺陷小鼠中有效恢复了蛋白质表达和视锥细胞功能。在本研究中，我们评估了这种基于慢病毒的疗法（LV-RPE65）在健康非人灵长类动物（NHP）上的眼部和全身耐受性，未进行辅助性全身抗炎预防。我们首次使用多模态成像描述了5只NHP视网膜下注射后2天、4天和7天视网膜脱离的早期动力学。我们发现，与注射载体的眼睛相比，注射LV-RPE65的眼睛视网膜重新附着时间延长。低剂量（n = 2）和高剂量（n = 2）LV-RPE65注射的眼睛黄斑区外核层和光感受器外节层厚度减小，比注射载体的眼睛（n = 4）更明显。所有注射LV-RPE65的眼睛均出现最初的血管周围反应，在14天内自发消退。尽管存在黄斑中心凹结构变化，但全视野视网膜电图显示，随着时间的推移，整体视网膜功能得以保留，免疫组织化学显示，低剂量、高剂量和注射载体的眼睛之间，神经胶质细胞、小胶质细胞或白细胞的眼部激活没有差异。此外，注射LV-RPE65的动物未显示载体脱落或眼外靶向的迹象，证实了载体在眼部的安全局限。我们的结果表明，在没有辅助抗炎预防的情况下，视网膜下注射LV-RPE65后眼部耐受性有限，与这种给药途径相关的并发症需要阻止这种短暂的炎症事件。
