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Eye-related diseases, specifically retinal dystrophy (RD) conditions, are the leading cause of blindness worldwide. Gene addition, regulation, or editing could potentially treat such diseases through gene expression regulation. CRISPR/Cas9 gene editing is one of the most prominent and precise gene editing tools which could be employed to edit genes related to the dystrophic condition. However, CRISPR/Cas9 faces in vivo delivery challenges due to its high molecular weight, negative charge, prone to degradation in the presence of nucleases and proteases, poor cellular degradation, etc., which makes it challenging to adopt for therapeutic applications. We developed cRGD-modified lipopolymeric nanoplexes loaded with Cas9 RNPs with a particle size and zeta potential of 175 ± 20 nm and 2.15 ± 0.9 mV, respectively. The cRGD-modified lipopolymeric nanoplexes were stable for 194 h and able to transfect >70 % ARPE-19 and NIH3T3 cells with an Indel frequency of ~40 % for the VEGF-A gene. The cRGD-modified lipopolymeric nanoplexes found good vitreous mobility and could transfection retinal cells in vivo after 48 h of intravitreal injection in Wistar Rats. Moreover, in vivo VEGFA gene editing was ~10 % with minimal toxicities. Collectively, the cRGD-modified lipopolymeric nanoplexes were found to have extreme potential in delivering CRISPR/Cas9 RNPs payload to the retinal tissues for therapeutic applications., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2024 Elsevier B.V. All rights reserved.)

CRISPR/Cas9 has proven its accuracy and precision for gene editing by making a double-strand break at the predetermined site. Despite being a mainstream gene editing tool, CRISPR/Cas9 has limitations for its in vivo delivery due to the physico-chemical properties such as high molecular weight, supranegative charge, degradation in the presence of nucleases, etc. Hereby, a cationic lipopolymer is explored for its efficiency in delivering CRISPR/Cas9 plasmid (pCas9) in vitro and in vivo. The lipopolymer is utilized to form blank cationic nanoplexes having a zeta potential of +15.8 ± 0.7 mV. Being cationic, the blank nanoplexes are able to condense the pCas9 plasmid at a ratio of 1:20 with a complexation efficiency of ≈98% and show a size and zeta potential of ≈141 ± 16 nm and 4.2 mV ± 0.7, respectively. The pCas9-loaded nanoplexes show a transfection efficiency of ≈69% in ARPE-19 cells and show ≈22% of indel frequency, indicating the successful translation of Cas9 protein and guide RNA in the cytosol. Further, they are found to be stable under in vivo environment when given intravenously in Swiss albino mice. These lipopolymeric nanoplexes can be a potential carrier for CRISPR plasmids for genome editing applications., (© 2023 Wiley-VCH GmbH.)