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.

中文翻译：

---

CRISPR 介导的 VEGFA 基因组消融治疗非人灵长类动物眼脉络膜新生血管的安全性和有效性


基于 CRISPR 的基因组编辑能够永久抑制血管生成因子，例如血管内皮生长因子 （VEGF），作为脉络膜新生血管形成 （CNV） 的潜在治疗方法，脉络膜新生血管是年龄相关性黄斑变性失明的主要原因。我们之前设计了具有化脓性链球菌 Cas 9 （SpCas9） 和向导 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，成功抑制了小鼠中的 VEGF 和激光诱导的 CNV。在这里，我们将平台推进到非人灵长类动物，发现与没有 gRNA 的 SpCas9 相比，具有靶向 VEGFA 的 gRNA 的视网膜下 AAV8-SpCas9 可能会降低 VEGF 和 CNV 的严重程度。然而，无论 gRNA 是否存在，所有接受 AAV8-SpCas9 的眼睛都出现了中心凹下沉积、同心性黄斑环和视网膜外破裂，这些在较高剂量下恶化。免疫组化显示视网膜色素上皮细胞、胶原蛋白和波形蛋白在中心凹下积累，光感受器结构破坏，视网膜神经胶质细胞和小胶质细胞活化。视网膜下 AAV8-SpCas9 触发了 CCL2 房水升高，但对 AAV8、SpCas9 或 GFP 报告基因的全身体液或细胞反应最小。我们的研究结果表明，CRISPR 介导的非人灵长类动物眼中的 VEGFA 消融可能会抑制 VEGF 和 CNV，但也可能导致意想不到的视网膜下纤维化、光感受器损伤和视网膜炎症，尽管全身免疫反应很小。