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.
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The main data supporting the results in this study are available within the paper and its Supplementary Information. Source data for the figures are available at Figshare (https://doi.org/10.6084/m9.figshare.12611819)76. The deep-sequencing and Nanopore DNA-sequencing data are available at the NCBI BioProject under the identifiers PRJNA642029, PRJNA593168 and PRJNA628164.
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We thank W. Yang at the Southern Medical University, China, for discussions and scientific input for the immunology part of the study. Y.C. is supported by the National Natural Science Foundation of China (no. 31971364), Pujiang Talent Project of Shanghai (no. 18PJ1404500), Shanghai Municipal Natural Science Foundation (no. 18ZR1419300), and startup funding from Shanghai Center for Systems Biomedicine, Shanghai Jiao Tong University (no. WF220441504).
The authors declare no competing interests.
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Ling, S., Yang, S., Hu, X. et al. Lentiviral delivery of co-packaged Cas9 mRNA and a Vegfa-targeting guide RNA prevents wet age-related macular degeneration in mice. Nat Biomed Eng 5, 144–156 (2021). https://doi.org/10.1038/s41551-020-00656-y
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