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Genome editing and knockout by virus-based delivery of CRISPR/Cas9 may provide a new option to cure inherited and acquired ocular diseases. Here we describe development and application of lentivirus-based delivery vectors enabling knockout of the Vegfa gene. We show that Streptococcus pyogenes (Sp) Cas9 and single-guide RNAs (sgRNAs) delivered by such vectors selectively can ablate the vascular endothelial growth factor A (Vegfa) gene in mouse retina following a single administration. These findings may contribute to the development of a new therapeutic path in the treatment of ocular diseases including exudative age-related macular degeneration (AMD).

Generation of lentivirus (LV)-based vectors holding multiple gene cassettes for coexpression of several therapeutic factors provides potent tools in both gene delivery studies as well as in gene therapy. Here we describe the development of such multigenic LV gene delivery vectors enabling cell-specific coexpression of antiangiogenic microRNA (miRNA) and protein factors and, if preferred, a fluorescent reporter, from RNApol(II)-driven expression cassettes orientated in a back-to-back fashion. This configuration may contribute to the development of new combination therapies for amelioration of diseases involving intraocular neovascularization including exudative age-related macular degeneration (AMD).