Therapeutic genome editing requires effective and targeted delivery methods. The delivery of Cas9 mRNA using adeno-associated viruses has led to potent in vivo therapeutic efficacy, but can cause sustained Cas9 expression, anti-Cas9 immune responses and off-target edits. Lentiviral vectors have been engineered to deliver nucleases that are expressed transiently, but in vivo evidence of their biomedical efficacy is lacking. Here, we show that the lentiviral codelivery of Streptococcus pyogenes Cas9 mRNA and expression cassettes that encode a guide RNA that targets vascular endothelial growth factor A (Vegfa) is efficacious in a mouse model of wet age-related macular degeneration induced by Vegfa. A single subretinal injection of engineered lentiviruses knocked out 44% of Vegfa in retinal pigment epithelium and reduced the area of choroidal neovascularization by 63% without inducing off-target edits or anti-Cas9 immune responses. Engineered lentiviruses for the transient expression of nucleases may form the basis of new treatments for retinal neovascular diseases.

治疗性基因组编辑需要有效且有针对性的递送方法。使用腺相关病毒递送Cas9 mRNA 已导致有效的体内治疗效果，但可能导致持续的Cas9表达、抗 Cas9 免疫反应和脱靶编辑。慢病毒载体已被设计用于递送瞬时表达的核酸酶，但缺乏其生物医学功效的体内证据。在这里，我们表明，化脓性链球菌 Cas9 mRNA 的慢病毒协同传递和编码靶向血管内皮生长因子 A ( Vegfa )的引导 RNA 的表达盒在由Vegfa诱导的湿性年龄相关性黄斑变性的小鼠模型中是有效的. 单次视网膜下注射工程慢病毒可敲除视网膜色素上皮中 44% 的Vegfa，并将脉络膜新生血管面积减少 63%，而不会引起脱靶编辑或抗 Cas9 免疫反应。用于核酸酶瞬时表达的工程慢病毒可能构成视网膜新生血管疾病新疗法的基础。