Robust aptamer-targeted CRISPR/Cas9 delivery using mesenchymal stem cell membrane –liposome hybrid: BIRC5 gene knockout against melanoma

In this study, a platform was fabricated by combining a cationic lipid, 1,2-Dioleoyl-3-trimethylammonium-propane (DOTAP) with mesenchymal stem cell membrane (MSCM) to produce a positively charged hybrid vesicle. The prepared hybrid vesicle was used to condense BIRC5 CRISPR/Cas9 plasmid for survivin (BIRC5) gene editing. The Sgc8-c aptamer (against protein tyrosine kinase 7) was then attached to the surface of the prepared NPs through electrostatic interactions. In this regard, melanoma cancer cells (B16F0 cell line) overexpressing PTK7 receptor could be targeted. Investigations were conducted on this system to evaluate its transfection efficiency, cellular toxicity, and therapeutic performance in preclinical stage using B16F0 tumor bearing C57BL/6 J mice. The results verified the superiority of the Hybrid/ BIRC5 compared to Liposome/ BIRC5 in terms of cellular toxicity and transfection efficiency. The cells exposure to Hybrid/BIRC5 significantly enhanced cytotoxicity. Moreover, cells treated with Apt-Hybrid/BIRC5 showed higher anti-proliferation activity toward PTK7-positive B16F0 cancer cells than that of the PKT7-negative CHO cell line. The active tumor targeting nanoparticles increased the cytotoxicity through down-regulation of BIRC5 expression as confirmed by Western blot analysis. In preclinical stage, Apt-Hybrid/BIRC5 showed remarkable tumor growth suppression toward B16F0 tumorized mice.

Thus, our study suggested that genome editing for BIRC5 through the CRISPR/Cas9 system could provide a potentially safe approach for melanoma cancer therapy and has great potential for clinical translation.