Sin Tzu-Ni, Tng Nicole, Dragoli Jack, Ramesh Kumar Sruthi, Villafuerte-Trisolini Carol, Chung Sook Hyun, Tu Lien, Le Sophie M, Shim Jae Ho, Pepple Kathryn L, Ravindran Resmi, Khan Imran H, Moshiri Ala, Thomasy Sara M, Yiu Glenn
Department of Ophthalmology & Vision Sciences, University of California Davis, Davis, CA 95616, USA.
Department of Surgical & Radiological Sciences, University of California Davis, Davis, CA 95616, USA.
Mol Ther. 2024 Sep 27. doi: 10.1016/j.ymthe.2024.09.027.
CRISPR-based genome editing enables permanent suppression of angiogenic factors such as vascular endothelial growth factor (VEGF) as a potential treatment for choroidal neovascularization (CNV)-a major cause of blindness in age-related macular degeneration. We previously designed adeno-associated viral (AAV) vectors with S. pyogenes Cas 9 (SpCas9) and guide RNAs (gRNAs) to target conserved sequences in VEGFA across mouse, rhesus macaque, and human, with successful suppression of VEGF and laser-induced CNV in mice. Here, we advanced the platform to nonhuman primates and found that subretinal AAV8-SpCas9 with gRNAs targeting VEGFA may reduce VEGF and CNV severity as compared with SpCas9 without gRNAs. However, all eyes that received AAV8-SpCas9 regardless of gRNA presence developed subfoveal deposits, concentric macular rings, and outer retinal disruption that worsened at higher dose. Immunohistochemistry showed subfoveal accumulation of retinal pigment epithelial cells, collagen, and vimentin, disrupted photoreceptor structure, and retinal glial and microglial activation. Subretinal AAV8-SpCas9 triggered aqueous elevations in CCL2, but minimal systemic humoral or cellular responses against AAV8, SpCas9, or GFP reporter. Our findings suggest that CRISPR-mediated VEGFA ablation in nonhuman primate eyes may suppress VEGF and CNV, but can also lead to unexpected subretinal fibrosis, photoreceptor damage, and retinal inflammation despite minimal systemic immune responses.
基于CRISPR的基因组编辑能够永久抑制血管生成因子，如血管内皮生长因子（VEGF），作为治疗脉络膜新生血管（CNV）的潜在方法，CNV是年龄相关性黄斑变性失明的主要原因。我们之前设计了携带化脓性链球菌Cas9（SpCas9）和引导RNA（gRNA）的腺相关病毒（AAV）载体，以靶向小鼠、恒河猴和人类VEGFA中的保守序列，成功抑制了小鼠体内的VEGF和激光诱导的CNV。在此，我们将该平台推进到非人灵长类动物，发现与不含gRNA的SpCas9相比，携带靶向VEGFA的gRNA的视网膜下AAV8-SpCas9可能会降低VEGF水平并减轻CNV的严重程度。然而，所有接受AAV8-SpCas9的眼睛，无论是否存在gRNA，都会出现黄斑下沉积物、同心黄斑环和外层视网膜破坏，且在高剂量时情况会恶化。免疫组织化学显示黄斑下视网膜色素上皮细胞、胶原蛋白和波形蛋白积聚，光感受器结构破坏，以及视网膜神经胶质细胞和小胶质细胞激活。视网膜下AAV8-SpCas9引发了CCL2在房水中的升高，但对AAV8、SpCas9或绿色荧光蛋白报告基因的全身体液或细胞反应极小。我们的研究结果表明，在非人灵长类动物眼中，CRISPR介导的VEGFA基因敲除可能会抑制VEGF和CNV，但尽管全身免疫反应极小，也可能导致意外的视网膜下纤维化、光感受器损伤和视网膜炎症。
