Factors Impacting Efficacy of AAV-Mediated CRISPR-Based Genome Editing for Treatment of Choroidal Neovascularization
Factors Impacting Efficacy of AAV-Mediated CRISPR-Based Genome Editing for Treatment of Choroidal Neovascularization
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DOI：
10.1016/j.omtm.2020.01.006
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发表时间：
2020-06-12
影响因子：
4.7
通讯作者：
Yiu, Glenn
中科院分区：
文献类型：
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作者：
Chung, Sook Hyun;Mollhoff, Iris Natalie;Yiu, Glenn
频繁注射抗血管内皮生长因子（抗VEGF）药物是新生血管性年龄相关性黄斑变性（AMD）患者的临床负担。使用成簇的规则间隔短回文重复序列（CRISPR）-Cas9的腺相关病毒（AAV）递送的VEGF-A的基因组破坏具有永久抑制异常血管生成的潜力，但是确定最佳功效的因素是未知的。在这里，我们研究了两种广泛使用的Cas9核酸内切酶SpCas 9和SaCas 9，并评估了体内AAV递送效率和基因组编辑速率的相对贡献，以确定驱动成功的基于CRISPR的VEGF-A抑制的机制，使用激光诱导的脉络膜新生血管（CNV）的小鼠模型。我们发现SpCas 9表现出比SaCas 9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SpCas 9的双载体方法和SaCas 9的单载体系统之间的AAV转导效率相似，以递送Cas9直系同源物和单向导RNA（gRNA）。我们的研究结果表明，使用AAV介导的CRISPR系统成功敲除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SpCas 9可能比SaCas 9更有效地作为基于CRISPR治疗新生血管性AMD中CNV的潜在治疗策略。
Frequent injections of anti-vascular endothelial growth factor (anti-VEGF) agents are a clinical burden for patients with neo-vascular 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.