Frequent injections of anti-vascular endothelial growth factor (anti-VEGF) agents are a clinical burden for patients with neovascular age-related macular degeneration (AMD). Genomic disruption of VEGF-A using adeno-associated viral (AAV) delivery of clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 has the potential to permanently suppress aberrant angiogenesis, but the factors that determine the optimal efficacy are unknown. Here, we investigate two widely used Cas9 endonucleases, SpCas9 and SaCas9, and evaluate the relative contribution of AAV-delivery efficiency and genome-editing rates in vivo to determine the mechanisms that drive successful CRISPR-based suppression of VEGF-A, using a mouse model of laser-induced choroidal neovascularization (CNV). We found that SpCas9 demonstrated higher genome-editing rates, greater VEGF reduction, and more effective CNV suppression than SaCas9, despite similar AAV transduction efficiency between a dual-vector approach for SpCas9 and single-vector system for SaCas9 to deliver the Cas9 orthologs and single guide RNAs (gRNAs). Our results suggest that successful VEGF knockdown using AAV-mediated CRISPR systems may be determined more by the efficiency of genome editing rather than viral transduction and that SpCas9 may be more effective than SaCas9 as a potential therapeutic strategy for CRISPR-based treatment of CNV in neovascular AMD.

中文翻译：

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影响 AAV 介导的基于 CRISPR 的基因组编辑治疗脉络膜新生血管疗效的因素。

频繁注射抗血管内皮生长因子（抗 VEGF）药物对于新生血管性年龄相关性黄斑变性（AMD）患者来说是一种临床负担。使用腺相关病毒 (AAV) 递送成簇规则间隔短回文重复序列 (CRISPR)-Cas9 对 VEGF-A 进行基因组破坏，有可能永久抑制异常血管生成，但决定最佳功效的因素尚不清楚。在这里，我们研究了两种广泛使用的 Cas9 核酸内切酶 SpCas9 和 SaCas9，并评估了体内 AAV 递送效率和基因组编辑率的相对贡献，以确定使用小鼠成功驱动基于 CRISPR 抑制 VEGF-A 的机制激光诱导脉络膜新生血管（CNV）模型。我们发现 SpCas9 表现出比 SaCas9 更高的基因组编辑率、更大的 VEGF 减少和更有效的 CNV 抑制，尽管 SpCas9 的双载体方法和 SaCas9 的单载体系统之间传递 Cas9 直系同源物和单载体的 AAV 转导效率相似。引导 RNA (gRNA)。我们的结果表明，使用 AAV 介导的 CRISPR 系统成功敲低 VEGF 可能更多地取决于基因组编辑的效率而不是病毒转导，并且 SpCas9 可能比 SaCas9 更有效，作为基于 CRISPR 治疗新生血管 CNV 的潜在治疗策略AMD。