Effective gene editing of melanoma by delivery of Cas9 mRNA with highly branched poly(β-amino ester)

Abstract

Melanoma, the most aggressive form of skin cancer, still lacks clinically reliable treatments. CRISPR/Cas9 gene editing offers a promising approach for melanoma therapy; however, the efficient and safe delivery of CRISPR/Cas9 components to melanoma cells remains a formidable challenge. Here, we report the development of highly branched poly(β-amino ester) (HPAE) for the efficient delivery of Cas9 mRNA to melanoma cells and for transdermal application in vivo. HPAE is synthesized via a one-pot “A2 + B4 + C2” type Michael addition strategy and is capable of efficiently condensing mRNA to form nanosized polyplexes with a moderate positive charge. In human embryonic kidney cells (HEK293T), mouse melanoma cells (B16F10), and human melanoma cells (A2058 and A375), up to 76.3%, 74.6%, 16.5%, and 57.1% mRNA transfection efficiency was achieved without obvious cytotoxicity. Moreover, repeated dosing further enhanced mRNA transfection efficiency, resulting in sustained gene expression. Importantly, HPAE enables the effective co-delivery of Cas9 mRNA and sgRNA to melanoma cells, facilitating up to 25.5% gene knockout. HPAE also demonstrates strong transdermal capability, resulting in high in vivo mRNA expression following direct topical administration. This study demonstrates the feasibility of treating melanoma through the transdermal application of the CRISPR/Cas9 system, enabled by the efficient mRNA delivery provided by HPAE.