Targeted knockout of the Vegfa gene in the retina by subretinal injection of ribonucleoprotein complexes containing Cas9 protein and chemically modified sgRNAs

Abstract The therapeutic effect of retinal gene therapy using CRISPR/Cas9-mediated genome editing and knockout applications is dependent on efficient and safe delivery of gene-modifying tool kits. Recently, transient administration of sgRNAs and SpCas9 proteins delivered as ribonucleoproteins (RNPs) has provided potent gene knockout in vitro. To improve efficacy of CRISPR-based gene therapy, we delivered RNPs containing SpCas9 protein complexed to chemically modified sgRNAs (msgRNAs). In K562 cells, msgRNAs significantly increased indel frequency (25%) compared to unmodified counterparts leading to robust knockout of the Vegfa gene encoding vascular endothelial growth factor A (96% indels). Likewise, in HEK293 cells, lipoplexes containing varying amounts of RNP and eGFP mRNA showed efficient Vegfa knockout (43% indels) and strong eGFP expression, indicative of efficacious functional knockout using low RNP amounts. In mice, subretinal injections of equivalent lipoplexes yielded 6% indels in Vegfa of isolated eGFP-positive RPE cells. However, signs of toxicity following delivery of lipoplexes containing high amounts of RNP were observed. While the mechanism resulting in the varying efficacy remains to be elucidated, our data suggest that a single subretinal injection of RNPs carrying msgRNAs and SpCas9 induces targeted retinal indel formation, thus providing a clinically relevant strategy relying on nonviral delivery of short-lived nuclease activity.